text:
The life of a college can be measured both by the success of its graduates and in its physical growth.
Seneca College has graduated thousands of students since its inception in 1967, and we have proudly seen them go on to be successful executives, senators, pilots, fashion designers, police officers, broadcasters and entrepreneurs.
A Sampling of Successful Seneca Alumni:
- Marc Caira — former President & CEO of Tim Hortons Inc.
- Hon. Alvin Curling — former MPP Scarborough North, Speaker of the Legislative Assembly, Ambassador to the Dominican Republic
- Armand La Barge — Chief of York Regional Police (retired)
- Vivienne Poy — Senator
- Philip Sparks — Fashion Designer
Building Through the Years
The Seneca of today was a long time coming. When the College first opened its doors in 1967, classes were offered at a number of sites including a former Woolworth store and a renovated factory.
Ground was turned in 1968 to create Seneca's first permanent location on Finch Avenue East at Highway 404. The modest building grew in phases over the years and is today named after our founding president William Newnham.
At the same time, Seneca created its Buttonville Campus to house its aviation program. Since then, the College's fleet has grown to include a total of 19 planes (single and twin engine), as well as simulators that allow students to gain experience in single engine planes and multi-crew jet aircrafts.
The 700-acre King Campus, the former estate owned by the Eaton Family, followed in 1971.
Recent Years
Many other locations have housed Seneca programs but the next major expansion happened in 1999 with the opening of the Seneca@York Campus, the first college campus to be located on the grounds of a university.
In 2005, Seneca opened its Markham Campus. A former 10-storey office building, this unique campus is located in Markham's business district, home to the head offices of many multinational and national businesses.
Seneca also has smaller campuses in Newmarket, Yorkgate and Vaughan offering community-based services.
In 2011, Newnham Campus celebrated the opening of the environmentally friendly A+ Building, providing a new entrance to the building and additional classrooms, study and meeting areas.
Students of the Bachelor of Aviation Technology program began classes at the Peterborough Aviaton Campus in January 2014. The new campus was created after the owners of the Buttonville Airport, where Seneca's aviation program had been based for more than 40 years, announced their intent to close the airport. The new state-of-the-art campus is located at the Peterborough Municipal Airport.
The Future
King Campus was awarded provincial funding to revitalize the campus by modernizing current facilities and building more space for students while expanding programming in health and community services, pathways and natural environment and sustainability. The $100+ million project to expand infrastructure at the Campus is made possible through a partnership with the Ontario government, Seneca Student Federation and the Student Athletic Association. Construction has already started on the 200,000 square foot Magna Hall, named in recognition of the $3-million donation to the Campaign for King Campus made by Magna International. It will feature 25 classrooms, labs, a library, a student centre, gymnasium and fitness facilities and provide space for 1,500 additional students.
At Newnham Campus, Seneca has broken ground on the Centre for Innovation, Technology and Entrepreneurship (CITE).
CITE, financially supported by the federal and provincial governments, will bring applied research and commercialization, specialized training and both student and business-led entrepreneurial activities under one roof.
It will include classrooms, labs and a presentation gallery that incorporates indigenous design. There will also be space and entrepreneurial supports to help small-to-medium-size enterprises from North Toronto and York Region build their innovation capacity through access to the facility's maker spaces, labs, equipment and research expertise.
Construction has also started, on a new multi-purpose artificial turf field and seasonal dome at Newnham Campus. It will serve both recreation and varsity programs, Seneca Summer Camps and the community through space rentals. It was made possible by funding from the Student Athletic Association and through the support of the Canada 150 Community Infrastructure Program.


text: List of President Trump's Accomplishments- Growing by the Day Economy 1. Passage of the tax reform bill providing $5.5 billion in cuts and repealing the Obamacare mandate. 2. Increase of the GDP above 3 percent. 3. Creation of 1.7 million new jobs, cutting unemployment to 4.1 percent. 4. Dow Jones at record highs. 5. A rebound in economic confidence to a 17-year high. 6. A new executive order to boost apprenticeships. 7. A move to boost computer sciences in Education Department program...More

subset: commoncrawl


text:
Once a year GiG runs a showcase event at their Malta office. It serves as an opportunity to take customers and gaming industry experts into the world of GiG, demonstrating new platform technology and celebrating the company's growth. For the 2017 event, FX were asked to create a VR application to immerse delegates in the beautiful scenery of Malta, highlighting tourist sites and GiG's history on the island.
We designed and built a 360° virtual reality application for the Samsung Gear. Displayed through Samsung Galaxy S8s, the application itself takes users on a journey through the sights of Malta.
Armed with footage from seven drone-mounted GoPros – supplied by 106 comms – the FX team stitched the sequences together and arranged them into a single 360° video.
Each shot was colour corrected for consistency within the experience, and to accentuate the natural beauty of the archipelago. By emphasising the Mediterranean weather and bringing out the highlights and richness of the sea we were able to add even more depth to the journey. Several cuts of footage were also created to show each of the main locations on the island.
Built in the popular game development platform, Unity, we were able to construct a high-quality virtual experience that would benefit from the versatility of the platform itself. Unity was also chosen for its ability to export to multiple formats, giving GiG the opportunity to offer a device-agnostic experience later in the project lifecycle.
By taking the composited 360° footage and mapping it onto the inside of a sphere we were able to place a VR camera rig inside the environment to represent the user's point of view. This technique works well in creating immersive experiences using 360° footage, with users given complete freedom to look around and explore the scene .
In order for the experience to feel branded and complete we integrated several bespoke UI elements, going so far as to create a 3D interactive map stylised to match GiG's neon colour pallet. And as we knew the application was going to be run at a busy event we crafted a simple control interface so as not to overwhelm users with multiple options that could potentially break the immersion. We opted for gaze controls, which enable the Samsung Gear to track the direction of a user's neck and head, translating them into a virtual environment. It's also one of the most natural control mechanics without the need to introduce any hand-held devices.
FX also created interactive hotspots inside the experience to allow users to discover more information about points of interest via text-based overlays.
The VR app was a great success. It became a key engagement driver for GiG throughout the event, drawing crowds of people while creating a great conversation piece between the business and its audience. The experience is now being rolled out at GiG's forthcoming events and engagement levels continue to increase.
Looking to increase customer engagement through VR? Or want to find out more about immersive media and the best approach for your business? We'd love to have a chat.


text:
Fashion forward moms
Sunday, May 12 is Mother's Day and on this day children and spouses put moms at the centre of attention by celebrating them in their own particular way, by showering them with love and appreciation.
Gifts of various kinds and a day out with mom are high on the agenda. Hitting the road with mom can range from a casual event to something more formal in the day or evening, depending on what you you think is appropriate for this special woman.
Of course, fashion is very important when it comes to celebrating a special day and Mother's Day is no different. So, just for you mothers out there, if you know or have any form of inclination of where you will be going on Sunday, we have some fashionable pieces just for you.
We turned to mother and daughter(s) teams to show just how fashionable both can be regardless of age and personal taste.
Happy Mother's Day all.
Suzanne Singh
Brittany Singh
Chelsea Singh
Alessandra Misir
Sara Misir
Clothing and accessories provided by:
Flirt Boutique
184 Constant Spring Road, Store 12, Manor Park Plaza. Telephone: 931-9332.
You can also find them on Twitter: @flirtboutique, Instagram: @flirtboutiqueja and facebook.com/flirtboutiqueja


text:
For many decades, Ayn Rand has been an icon of the libertarian right in the USA. Novelist, philosopher, economist, commentator, critic, she argued for what became known as "Objectivism". In a nutshell (and if I've understood it correctly), she argued that if we all stick to the knitting and plough our own furrow, then everyone concerned — we ourselves, our associates, customers, supply chains and society in general — will do better than if we all go round deferring to the interests of third parties, and, by implication, put our own interests second. Or to quote her own book title, she believed in "The Virtue of Selfishness".
Perhaps those who agonise today about "Corporate Social Responsibility" would do well to read her work. A corporation discharges its social responsibility by providing goods and services, creating jobs, paying wages and pensions, and paying taxes — not by running youth clubs and charities.
Ayn Rand's greatest work was her last and longest novel, "Atlas Shrugged" . Just about every libertarian undergrad you meet in America will have read it.
So I was ashamed that I myself had never read it. But I was gratified, during the holiday month of August, that at least I made a start. It's a big project. I have the Signet anniversary edition from 2007, given me a couple of years ago by an American intern in Brussels. It's in paperback. It's in what looks like about eight-point type, densely packed. No pictures! And it runs to 1067 pages. It's a bit daunting as a holiday read.
So far I'm at about page 200 or so. But already I've come across passages that resonate very loudly in 2015. The story centres on a young woman corporate executive called Dagny Taggart. (Whoever heard of a girl called Dagny? Come to that, whoever heard of a girl called Ayn?). She runs a railway, and is determined to use an innovative alloy in place of steel for her rails and bridges. So of course she meets massive resistance from the entrenched makers and users of steel. There's a vicious smear campaign against the new technology, with rail unions threatening to boycott it. It reminded me of the hysterical propaganda we're seeing today against shale gas.
Two particular passages stick in my mind. The CEO of the company making the alloy is visited by a slightly sinister bureaucrat from the "State Science Institute". He has no evidence against the new alloy, but he comes up with phrases like "The State Science Institute does not hold a favourable opinion of {the alloy}". And a little later, "The State Science Institute represents the best brains of the country, Mr. CEO". When I read those words, I thought immediately and with great affection of the IPCC. Heaven knows why.
Our heroine Dagny then goes to see the head of the State Scientific Institute. He prevaricates for a long time, but finally admits that he has not read his own Institute's report, and accepts that he knows nothing to the detriment of the new alloy. But he finally admits that has come under pressure from legislators, who in turn are responding to the interests of existing industry players who are threatened by the innovation. He fears that he will lose the funding for his Institute unless he publishes a report rubbishing the new alloy.
Ayn Rand had an extraordinary premonition of the way in which science can be corrupted by commercial and political interests. If she were alive today, she would be saddened but unsurprised by climate alarmism, and the way in which a small group of "experts" have been allowed to hi-jack the IPCC, and to cause many billions of dollars' worth of economic damage to be done to Western economies.
I look forward to reading the rest of Ayn Rand's masterwork (though heaven knows when I'll get time). She'd have been 110 this year, if she'd lived.


text:
Meiya All Naural Rubber Squeaker Boxed
Meiya the Mouse all natural rubber squeaker toy. Perfect for a baby or young child to chew on.
A part of the Meiya & Alvin collection which is an environmentally friendly range of toys from Bonikka. They are made with natural cotton, antibacterial corn fibre and sustainably produced rubber. These toys are hypo-allergenic and non-toxic, and are suitable for babies.
It's a good feeling to know that you are giving baby something that is produced with an environmental conscience, looking after baby and looking after the planet. A perfect purchase!


text:
Big Boi & Andre 3000 could have prevented Janet Jackson's nip slip.
The New England Patriots narrowly defeated the Carolina Panthers in Super Bowl XXXVIII, but no one remembers that. All that anyone outside New England remembers from that Super Bowl is when Justin Timberlake accidentally-on-purpose ripped of Janet Jackson's shirt and bared her titty for all of Earth to see. The "wardrobe malfunction" could have been prevented, if Outkast had accepted the NFL's invitation to perform at that year's Super Bowl.
In an interview with Dan Le Batard, Big Boi revealed that he and Andre 3000 had received an offer to perform at the Super Bowl XXXVIII halftime show, but they reached an impasse when Andre wanted to sing "Hey Ya" and "The Way You Move" and the NFL said no.
"He didn't want to cut the songs," Big Boi said. "He wanted to perform both songs in full and they didn't give us enough time to do it so we passed."
Fingers crossed that the NFL will one day come to its senses and invite them back. Listen to Big Boi's interview HERE.


text:
When: Wed., Sept. 26 2012
It was just last year when Stevie Stone hooked up with Tech N9ne's Strange Music label, but he's already immortalized his signing in song: "Really could feel it when the headlines read, Strange Met Stone," intones the Missouri-born emcee on "Perfect Stranger." "When they asked me how I feel, I said I'm finally home." (Awwww!) Stone's hoping the connection will land him a slot at next year's Gathering of the Juggalos, but in the meantime you can make him feel at home by smuggling a few liters of Faygo into the Black Sheep (2106 E. Platte Ave., blacksheep-rocks.com) for this 7 p.m., all-ages show. Tickets are $15 in advance, $25 at the door, with Flawless & CES CRU, 17 Ents, Big Cheez, Solo, Young Ghost and Dirty Gamez rounding out the bill. — Bill Forman


text:
- Automatic quantification of in vivo proton MR spectra.
- Some early user applications of LCModel can be seen in the sample publications list.
News:LCModel Version 6.3 (free upgrade for current LCModel sites):
- Estimation of GABA with MEGA-PRESS.
- Improved estimation of weaker metabolites, especially glutamine.
- Improved analyses of muscle, lipid, breast and liver spectra.
Past News:LCModel Version 6.2. While the main application remains brain, options have been added for more primitive spectra:
- Mainly water & lipids, as in fat and
- Water & lipids plus possibly choline, as in
breast and
- LCModel Version 6.1 with new
options, including analysis of muscle spectra,
particularly for IMCL.
- A Linux version for PCs (with Intel or AMD processors) is
now available. It is much faster than UNIX, with an excellent hardware
performance/price ratio, particularly interesting for MRSI.
- In Japan, you obtain LCModel (and support) directly
from the exclusive LCModel distributor for Japan, L.A. Systems,
- Major new LCModel Version 6
with many new features, including:
- Estimates of macromolecule and lipid contributions in normals and in pathologies (e.g., tumors) with strong lipid signals and without strong lipid signals.
- Analyses of full 2D slices or rectangular 2D subsets of 2D or 3D MRSI
data sets.
- Fully automatic, non-interactive, operator-independent: time-domain
data input, one-page summary output.
- Fully developed, over 28 years, with spectra analyzed from a wide variety of scanners and field strengths at more than 1000 sites.
Download, Install & Test:
- You can freely download the complete LCMgui/LCModel package for your Linux PC. (The two
commands needed for
installation and the run with the test data are also specified.) You can also
test the preparation and input of your own data to LCModel.
- You can fully enable your LCModel package to analyze all data (not just the test data) with a license.
Methods are described in:
- Stephen Provencher: Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn Reson Med 30, 672 (1993).
- User's Manual (you can always get the latest version here).
LCMgui Graphical User InterfaceThe normal usage is to supply the time-domain data as a simple text file to LCModel. A possible alternative is the LCMgui graphical user interface:
- Freely available for use with LCModel.
- Simplified usage, often only with mouse clicks, in two steps:
- Select the data with the File Selector;
- Start LCModel (optionally checking and modifying any settings) in the Control Window.
- Select the data with the File Selector;
- Currently for the following single-voxel data:
- Bruker
- GE 5.x Probe raw P-files and spectrum G-files.
- GE Probe raw P-files.
Also for P-files with multi-channel (phased-array) data.
- Hitachi data: part of the Hitachi software; no need to purchase
- Philips SDAT/SPAR pairs and enhanced DICOM files.
- Picker (later Marconi, now Philips) DUMP files.
- Siemens *rda files transferred from the syngo PC
console and
Siemens raw files from the (old) Numaris-3 Unix console.
- Canon/Toshiba rawData files and
Version 7.xx (and later) DICOM files.
- Varian/Agilent
- Other data types using your own conversion script.
- Bruker
- Currently for the following MRSI data:
- Philips, Siemens & Canon/Toshiba single-channel data,
including interactive display, selection and analysis of full 2D slices or
rectangular subsets. (Instructions in Sections 3.2 & 3.7 of the LCModel Manual.)
- Other data types using your own conversion script;
- For GE MRSI (and single-voxel) data, Mary
McLean's SAGE/LCModel interface [MA McLean et al, Magn Reson
44, 401 (2000)] is integrated into GE's SAGE
Research version (from SAGE Dev2002.1).
- Philips, Siemens & Canon/Toshiba single-channel data, including interactive display, selection and analysis of full 2D slices or rectangular subsets. (Instructions in Sections 3.2 & 3.7 of the LCModel Manual.)
Contact: Stephen Provencher: email@example.com
To Inverse Problems home page
Last modified: 22 November 2019


text:
Ranger-Tim Holtz distress re-inkers are the perfect way to rejuvenate your existing distress inkpads (sold separately)!
This glass bottle contains ½oz fluid reinker and an eye-dropper style applicator.
Can be used as a painting medium, simply dispense a little into a platic container then use a dry brush to apply to your project. Create shading effects using a damp brush.
Please note: This 'special order' item is not available for immediate dispatch, however, you may still order it by clicking on the 'special order' button above and we will dispatch it to you when is arrives from our supplier.
If the item is not already on order with our supplier then we will need to add it to our next order.
Providing our supplier has stock available it could take anywhere from 5 - 21 working days to arrive with us.
Thank you.
Copyright © 2002-2020 sirstampalot.co.uk
All Rights Reserved.


text:
Who has held the oceans in His hands?
Who has numbered every grain of sand?
Kings and nations tremble at His voice
All creation rises to rejoice
Behold our God seated on His throne
Come, let us adore Him
Behold our King! Nothing can compare
Come, let us adore Him!
Who has given counsel to the Lord?
Who can question any of His words?
Who can teach the One Who knows all things?
Who can fathom all His wondrous deeds?
Who has felt the nails upon His hands
Bearing all the guilt of sinful man?
God eternal humbled to the grave
Jesus, Savior risen now to reign!
Men: You will reign forever
Women: Let Your glory fill the earth
Music and Words by Jonathan Baird, Meghan Baird, Ryan Baird, and Stephen Altrogge © 2011 Sovereign Grace Worship (ASCAP)/Sovereign Grace Praise (BMI). Sovereign Grace Music, a division of Sovereign Grace Churches. All rights reserved. Administrated worldwide at www.CapitolCMGPublishing.com, excluding the UK which is adm. by Integrity Music, part of the David C Cook family. www.SovereignGraceMusic.org
Themes: God, Creator, Glory of God, Sovereignty, Jesus Christ, Reign of Christ
Scripture References: Isaiah 25:9, Isaiah 35:4, Isaiah 40:12-17, Psalms 46:6-10,
Liturgical Elements: Adoration
Related: Contempla a Dios,Eis nosso Deus
Translations:Chinese Croatian Dutch Farsi French Haitian Creole Korean Portuguese Russian Spanish Welsh
Featured ResourcesGuitar Chart - (C)Lead Sheet - (C)Piano Score - (C)
Album-specific Resources:
Chord Chart - Recorded Key (C)
Album Lead Sheet - Recorded Key (C)
Listen to a Sample
Guitar Chart - Recorded Key (C)
Listen to a Sample
Guitar Chart - Recorded Key (C)
Listen to a Sample
Guitar Chart - Recorded Key (C)
Guitar Chart - Alternate Key (E)
Lead Sheet - Alternate Key (E)
Piano Score - Recorded Key (C)
Piano Score - Alternate Key (E)
Download Orchestration (Andrews)


text:
Just a week after filing for divorce from her husband of two years, Kevin Federline, Britney Spears was seen having dinner with her son, Sean Preston, and a mystery man.
But despite reports of a new romance, the "mystery man" is none other than Brit's brother.
As Britney bounces back, we've discovered how Spears reached her breaking point in her marriage to K-Fed.
Us Weekly reports that Spears finally snapped after Federline sent her a handwritten letter blasting her for their problems.
"He blamed her for all of their problems, and that was when she said she had enough," dished the magazine's Katrina Szish.
The magazine also revealed that Britney's mom, Lynne, and the star's divorce attorney held an intervention demanding Britney drop kick K-Fed out of Malibu and out of her life.
Now, Us Weekly also reports that the pop princess' former flame, Justin Timberlake, may write a song for her comeback album as part of Britney's dramatic makeover.
And speaking of Brit's magical transformation, her hot new look is causing some to speculate.
Even Us Weekly suspects Ms. Britney may have had a little work done.
"Experts are telling us she may have had breast augmentation," Szish said.
But then again, it could also be the side effect of just giving birth to son Jayden James; Britney's rep declined to comment.
No matter what she's doing, the world is ready for Britney's sensational comeback.


text:
Enter your email address, password, and optional promo code below to log in to your account.
First time here? All museum members have accounts waiting for them—all you have to do is click Forgot your password? to set up your login (use the email associated with your membership). Not a member? Click here to register for a new account. Need help? Email firstname.lastname@example.com


text:
My Passion –
I am in love with technology, particulalry internet technology. My focus is on affordable consumer technologies right now. The idea that technology can be a equaliser and democratise access to information is quite heartening. I want to see how we can transform each others lives with technology.
My mission –
I am pained by the way Mumbai is turning out. Instead of getting rid of potholes, people prefer to buy costlier cars. Instead of having better medical care people prefer to opt for the expensive healthcare solution. The state is pretty much saying that if you can afford it, you can have the quality of life fit for a decent living. Otherwise you are pretty much on your own.
I want to make cities better. I want our cities to offer better houses, efficient transportation, and safe and vibrant neighbourhoods.
My Goals
Leverage Technology to make city better. Make an Impact, increase brand recognition and reach..
Photo by Dan Gribbin on Unsplash


text:
Wednesday, February 12, 2014
This is California Gulch. It's below California Pass. It's not in California. It's in Colorado. It's near Silverton, in the San Juans.
Monday, February 10, 2014
This is what fall looks like just outside of Crested Butte, Colorado. It's pretty nice.
This is a view of Dolores Peak and Woods Lake just after sunrise, in Uncompahgre National Forest, SW Colorado.
I just got Lightroom. Believe it or not, up until now I've been doing all of my image post-processing in Picasa. It's (very) basic, but assuming your original image is in pretty good shape, you can usually get some pretty good results.
But, I got Lightroom. And this is my first ham-fisted attempt at using it. The problem is, my computer is old. Very old. And Lightroom doesn't run very fast. Like, when I move a slider, it takes 4-5 seconds to see the result. This makes subtle adjustments just about impossible. (If you want to move a slider back and forth 4 or 5 times to get it just right, you'll be sitting there for a couple minutes – for ONE adjustment. Yarrghh.)
So, I'm basically looking at upgrading my PC now. And in the meantime, there's this one. I feel like it's a bit over-processed. Also, I tried starting from scratch a couple times to se if I could replicate the results (to see if I really understood what I was doing), and I couldn't come close (which means I don't, yet.) Oh well, figured I'd go ahead and toss it up here, since I had it and all.
Vermilion Peak from just below Hope Lake.
Wednesday, July 31, 2013
You're looking at Palmyra Peak (close on the left) and the Wilson Massif (Mt. Wilson, Wilson Peak and El Diente Peak) (far, on the
left right). You can also see Lizard Head, it's the little pokey thing a little bit right of center. This was taken from Telluride Ski Resort, near the top of the Revelation lift.


text:
Helloooo :) It was really nice for a couple days this past week, and I had to take advantage of the nice weather and do more outdoor outfit pics! I really enjoy nature and being outside, especially after this long winter of being cooped up inside because it was either a blizzard or it was below 0.
This week came and went in a flash. It's nearing the end of the semester and I'm honestly really happy because this one hasn't been the best for me.
I didn't have any creative classes OR fashion classes so I felt really uninspired by the work I am doing to get through these classes. I thought about retaking a writing class just to have a creative outlet. But, here we are....at the end. Phew. I'm wearing one of my favorite dresses and pair of flats, both from ModCloth. I really adore this dress because I really went out of my comfort zone with the plaid, but I've learned that it's really so versatile.
Glasses: Pierce from Warby Parker// Dress & Flats: ModCloth.com// Cardigan: JCP// Tights: Target
Follow my blog with Bloglovin
Follow my blog with Bloglovin


text:
Shocking Video Of Grandfather Dumping Ice On A 10 Month Old For Ice Bucket Challenge [VIDEO]
The Ice Bucket Challenge has been quite a success, as people from all walks of life take part in raising money and awareness about ALS. Unfortunately, some people take it too far, in this case a grandfather dumping ice on his ten month old granddaughter.
A ten month old is a baby and a should be treated as such. Dumping freezing ice on a baby is a horrible idea and I have no idea what this grandfather was thinking. Was it related to his daughter's nasty divorce? If so, a child should never be used like that. I'm appalled by this video. That poor baby must've been so shocked and cold.


text:
Maybe he has something else lined up.Why would he willingly jack in a secure,well paid job ?
Sounds to me that he was on a final warning and jumped before he was pushed.
Naturally, he gains bonus points for such a wokeful justification for withdrawing his labour.
Have I misunderstood what he said ?


text:
- 4 Concert Photography Tips
Lightchasers Photography
A good set of concert photography tips including lights, angles, expressions, and significant others.
- 10 Tips for Being a Greener Photographer
A few useful pointers on how to keep your photographic habits from smacking the environment around too much.
- Photo Projects – Why you should participate
digital Photography School
I love photography projects, and here are a ton of reasons why they're so great.
- May Challenge
The May Challenge is coming up, and Trevor has decided that we'll be shooting specific objects each week. He just hasn't told us which ones yet!
- 10 ways to make Google love your photography site
SEO tips for photographers — great advice to get the search engines looking at your images.
- The Impossible Art of Li Wei
The work of Chinese artist Li Wei from Beijing is a mixture between performance art and photography. See the photos here.
- Earth Day 2008
1001 Noisy Cameras
Earth Day roundup with 50+ stories!
- The Wall Street Journal lifted my image!
LeggNet's Digital Capture
Nice… Even the Wall Street Journal is lifting images from Flickr. Way to be a good example guys.
- Camera Raw, Bridge, or Lightroom?
Photoshop Insider
Scott Kelby goes over the similarities and differences between RAW processors found in ACR, Bridge, and Lightroom.
- Six and Two Halves Must-Read Free Digital Photography eBooks
A good collection of free eBooks on many different topics. Check them out — you're bound to find at least one of them useful.
- Orphan Works bill introduced in US
Have you heard about the Orphan Works legislation yet? If not, here's a good overview of what it is and how it works.
Tag Archives: stories
Link Roundup 04-19-2008
- Share the Love
Neil Creek
Neil has another great project starting up this month, and he's asking you to take a photo of something in your life that you love.
- Blog Statistics – Most Popular
Photography related bloggers and photo sharing enthusiasts are invited to share which of their articles/pictures were read/seen the most.
- Notes On Process
Joseph Szymanski
Notes from a film photographer on his darkroom process. From film to processing to enlarging to paper and developing — he covers the basics and puts us digital folks to shame.
- When Editorial Uses of Photos Require Model Releases
Dan Heller's Photography Business Blog
Model releases are always a big deal with stock photography, but even editorial photography can require them in certain instances. Here's a rundown on the rules.
- Ok, So Most of You Can Tell Graffiti From Fine Art
Thomas Hawk's Digital Connection
Thomas Hawk did a little experiment on his blog by presenting two paintings: one work of fine art and one piece of graffiti on a wall. Most guessed which was which, but I found myself more attracted to the graffiti (as did others).
- Buyer's guide: Buying a digital camera for your kids
John Watson goes through his process for buying a camera for kids. Lots of things to think about and lots of options out there.
- 10 Cameraphone Photographs that Rock
digital Photography School
Great photos from little cameras — It's pretty amazing what you can do with a camera phone.
- Photoshop Lightroom Workflow Part 1: Importing & Organizing
One photographer's approach to the digital workflow using Adobe Lightroom. Part 2 of this mini-series is yet to come.
- Seven Reasons to Share Your Photographic Know How Online
Sharing of knowledge is great — I'm a huge advocate of such things. Here's one photography blogger's take on sharing your know-how online.


text:
Miranda Kerr is very aware of her son; whenever her schedule permits.
As the Mango AW13-14 campaign; one year more...
Or the photos as 'Cicciolina' where she show much of her anatomy for V magazine.
Fine Miranda, a beauty session with friends always brings you back to the real world.
Source: @mirandakerr.


text:
This explanation of Easter, including both Christian beliefs and the various festive foods, as an imaginary conversation with a 4 year old is a lovely starting point for working out how to explain it to your children in a Christian household. Thank you Oh Baby magazine.
(I note that scholarly works, including Bede in 8th Century England, acknowledge that festive spring-time celebrations pre-date Christian celebrations of Easter in many northern European countries and elements of these were incorporated into Christian celebrations. It is believed that the hare (rabbit) and collecting eggs (or candies) are very old folk traditions / symbols that have morphed into something far more commercial and chocolatey in our present day than rabbit pie! I do like the explanation below that at its simplest can be that Jesus wants us to have fun and celebrate life, including Christ's resurrection, and, well, chocolate is awesome.)
"My nearly four-year-old is busy studying the latest flyer from one of the big stores, so conveniently deposited into our mailbox ever other day. This one is seasonally filled with chocolate eggs. Big ones, small ones, hollow ones, marshmallow ones, licensed ones featuring the latest cartoon star/movie star/pop star. She is dreaming, mouth almost watering. I am curious:
Hey darling, can you tell me what Easter is really about?
Ummm… Eggs?
No, not really.
Ummm… Chocolate?
Ummmm…. Chocolate eggs?!
No, not even chocolate eggs. Do you remember what happened at the first ever Easter?
Ummm… Jesus was born?
No, not quite. Jesus was born at Christmas time. He came into this world as a little baby, but he was actually God's son and God was sending him to this world because we needed a Saviour.
Why did we need a saviour?
Well, this world is pretty amazing and beautiful. But people do some dumb stuff sometimes. They hurt each other and that makes God really sad. They also forget about God and try and manage on their own. But God loves us so much, He doesn't want us to hurt each other, or ourselves, and He misses us if we just go off doing our own thing. So he came up with a plan. He would send his son Jesus to be with people on earth for a while, so they could learn more about God and the way God wants us to live. And then Jesus could take all the punishment for the wrong things people do, so us people – God's precious children, would not be separated from Him forever but could stay connected to God and one day live with Him in heaven for eternity.
What's eternity?
Forever and ever. But back to Easter. Jesus had been living on earth for about 33 years.
Daddy is going to be 33 at his next birthday!
Yep, Daddy is nearly 33. Jesus had been working as a carpenter…
What's a carpenter?
A carpenter is a builder.
A builder like Daddy!
Yes, a builder like Daddy. Jesus had been building, but also telling people amazing stories, and teaching them really helpful things about life, and making sick people better again, and performing miracles.
What's a miracle?
A miracle is something so amazing that only God could have made it happen.
Like me?
Yep, you're a miracle. But back to Easter. People were hearing about Jesus and all the cool stuff he was doing. And some people knew he was a great leader and they wanted to follow him. But other people were afraid of Jesus. They were worried that Jesus might make trouble for the rulers, that Jesus would get people to fight against their rule and try and take over.
But you said Jesus was only doing cool stuff?
Yes, he was. He wasn't going to fight with anybody. He was doing what God had asked him to do – teach people about the kingdom of God. But the really powerful rulers felt so threatened by Jesus that they got soldiers to arrest him, saying that Jesus had broken the laws of God. He hadn't broken any laws, but by now the priests had told lies about Jesus to the Governor. The Governor didn't want Jesus killed, but people were all getting really upset and out of control, so the Governor thought Jesus had to be punished and then hopefully people would calm down again.
Did Jesus get time out?
Well, sadly not. They did a really awful thing to Jesus. They nailed him to a big wooden cross, which is really, really bad for your body, and after a while Jesus couldn't breath anymore, and he died. And we remember that day on Good Friday. We have Hot Cross Buns, and the crosses remind us what happened to Jesus that day.
Good Friday? That cross thing doesn't sound very good.
Well, something really good was about to happen. And the cross thing – well, that was very, very important, because it was part of God's plan for Jesus. So it was a holy thing to happen. And another word for "holy" is "good".
Did Jesus stay on the cross?
No, his friends were allowed to come and take him down from the cross. They took his body and wrapped it up, and then they carried it to a tomb.
You mean a room?
No, a tomb… a tomb is like a cave in rock, and in those days that's where they put people when they had died. And then a big rock was rolled in front of the tomb and a guard was there keeping an eye on things.
Were his friends sad?
Yes, they were really, really sad. And they comforted each other. And then on the third day, the Sunday, some of Jesus' friends, some ladies, went up to his tomb.
To take Jesus some flowers?
Something like that. But do you know what they found? Nothing! No Jesus!! And the big huge heavy stone had been rolled away! The tomb was empty, and the guard had no idea what had happened. And then they saw a couple of angels who told them that Jesus was alive! He had come back to life, he had risen from the dead! We call that resurrection. That's why Easter Sunday is called Resurrection Sunday. The friends of Jesus were amazed, and ran back to their other friends to tell them the good news. It was hard to believe it was true, so Peter, another one of Jesus' friends came up to the tomb and all he could find were some of the bandages they had wrapped Jesus in before.
Jesus was alive, and he visited with his friends and talked to them some more before going up to Heaven to be with God.
Why did he go back to heaven?
Well, his work on earth was done.
His building work? Had he finished his house?
Not his building work, but the job God had sent him to earth to complete – Jesus had to die as punishment for our sins – the things we do wrong. Because Jesus took the punishment for us, we don't have to be punished, we can be forgiven and still stay really close to God, even though we make mistakes and do the wrong thing sometimes. But God's plan was not just Jesus dying on the cross, the most amazing part was that empty tomb. The empty tomb showed us God's amazing power – God is more powerful than death, so Jesus came back to life! God is the greatest, most powerful king and ruler EVER. And because of Jesus dying on Good Friday but then coming back to life on Resurrection Sunday, we too don't need to be afraid of dying – we can live forever with God. In fact, with God we don't need to be afraid of anything!
So… why do we have Easter eggs?
That's a good question. The hollow chocolate eggs, the ones with nothing inside, can remind us of that empty tomb… there was nothing inside the tomb because Jesus was alive!
Does Jesus know the Easter bunny?
He might do. Jesus is actually really into fun stuff, and people having a good time. The bible says he came so that we may have life, and life in abundance! That means a really fun, happy, full life! At Easter there is heaps of fun stuff around. Some things – like eggs and baby chickens represent new life, the new life we can all have because of what Jesus did. Other things are symbols of spring because in the Northern Hemisphere Easter happens at springtime. It's not Spring here in New Zealand though, its actually Autumn, but that's ok – we can still think about Spring and all the new life that happens then when animals are born and chickens hatch out of eggs…
So why does this Easter Egg here in the magazine have a picture of Dora on it?
That, my darling, I just can't really explain."
"For God so loved the world that he gave his one and only Son, that whoever believes in him shall not perish but have eternal life." John 3:16


text:
With the growing success of the hit song "Happy," by Pharrell Williams, the EventsDC staff joined the trend to showcase their own version of Pharrell's Oscar nominated hit. The video, which features staff and service partners across the official convention and sports authority's multiple properties, showcases a first hand look at just how happy the employees are to work at Events DC. The staff is excited to spread happiness all over Washington, DC and offer a glimpse into our engaging work environment.
See what makes our team Happy here:


text: Oh man! Ad-blocking software has been detected! :'( This website is run by the community, for the community... and it needs advertisements in order to keep running. Blocking our ads means your killing our stats! Please disable your ad-block, or become a premium member to hide all advertisements and this notice. i study with a training provider that not only condones but encourages the use of braindumps, which totally pisses me off. theres a "lab" which i never use because its too noisy, but on every computer there there are copies of test king, and thats all anyone ever does on those machines, just studies those questions. during class they will suggest certain braindump sites to use. one of the guys goes there every day and "studies" the test king files all day long. he's being doing that for a few months, and should be taking two exams soon, the 70-270 and the 70-290. i decided to ask him a simple question to see what he knew so i did an ipconfig on a pc and saw it had a 169.254.x.x IP. i showed it to him and asked what he could tell me from looking at that address and he was like blank. if all he knows is the answers from TK then i sincerely hope he fails. also i do all the hard work of actually studying while those losers will get qualified without ever knowing anything. all he will know is the questions from TK. put a twist on the questions and he'll be lost. i'm just a bit pissed right now.


text:
Far off in Eastern Anatolia
It's been a long day, so this will be but a brief update from the road.
We took an early morning flight across Turkey to the eastern city of Van. A remote outpost not far from the Iranian border, Van is a beautiful – if not harsh – place. And, it is one you may have heard of recently on the news, as just yesterday over 2000 people crowded onto Akdamar Island here to celebrate at the Akdamar church, an ancient Armenian church perched on a hilltop surrounded by brilliant, azure waters.
After a nice visit on Akdamar, we returned to Van and made a requisite stop at Van fortress. Another ancient edifice, the Van fortress was first built around the 7th century BC by the Urartians. Knocked down and rebuilt for centuries, crumbling walls and a new, towering minaret are all that remains of this proud building. Nonetheless, it is a stunning place, with views across the Van sea to the surrounding peaks…including Mount Artos, our objective for tomorrow.
Not a high peak, Artos stands a mere 11,604 feet high. But, it should be a good acclimatization run before heading Wednesday to Camp 1 on Ararat. Blue skies and a full moon are in the forecast, so things are looking good.
More to come soon!
-Jake Norton is an Everest climber, guide, photographer, writer, and motivational speaker from Colorado.


text:
What should I know about Asthma?
Asthma is a common lung condition in the industrialized world, one that impacts the lives of adults and children alike. Asthma threatens one's essential ability to breathe, to take in oxygen, our most basic and urgent survival need. We cannot live without air for more than a few minutes. Choke off our air supply, and we quickly become frightened and desperate, to the point of panic. It is easy to understand why asthma is one of the most taxing, debilitating health problems a human being can face, both physically and mentally.
The most significant characteristic of asthma is a narrowing of the trachea (the "windpipe") and bronchial passages in response to some stimulus. In asthma, the airways are over-responsive. Certain things that may not cause breathing problems for most of us can provoke the airways to constrict in asthmatics. These may be external stimuli like pollen, dust, and even cold air. Or, the stimulus may be something internal, like a nutritional deficiency or emotional stress. Whatever the trigger, all asthma attacks have the same scenario in common: the airways become extremely irritated and overreact by going into spasm. (1)
When the airways are constricted by an asthma attack, their delicate membrane linings become swollen and inflamed. Almost two-thirds of all people with asthma, about 65 percent, experience their first symptoms—wheezing, shortness of breath, and rapid heartbeat-- by age five. Although asthma is a chronic lung disease that persists for years and even for a lifetime, the bronchial constriction itself is reversible; the attack can be stopped and normal breathing restored.
Asthma is divided into two major types: "intrinsic" asthma and "extrinsic" asthma. Intrinsic asthma usually develops in adulthood and may begin with risk factors such as cold air, exercise, or emotional trauma. In extrinsic asthma, also called allergic or "atopic" asthma, the immune system reacts to an allergy-provoking substance such as pollen or dust by releasing histamine and other chemicals in the lungs. These potent chemicals cause smooth muscle in the air passages to secrete mucous, swell up, and narrow. The specific triggers of an asthma attack may be allergic or non-allergic.
Some causes of asthma attacks include: (2)
American Academy of Allergy, Asthma, and Immunology, 2005.
Wheezing, coughing, and difficulty breathing are the major symptoms associated with asthma. (3) The length and severity of asthma attacks can vary. (4) These symptoms are frequently triggered by upper respiratory tract infections (viral and bacterial), exercise, exposure to irritants (allergic, chemical, and physical), psychological problems, or climate. (5) , (6) Airway narrowing can sometimes occur before the symptoms of a full-blown attack. (7) People with wheezing, coughing, and trouble breathing at the same time should seek medical attention to rule out asthma, especially children who have had an upper respiratory infection.General
Long-term Preventative Medications:
Anti-inflammatory agents:
Anti-allergenic agents:
Quick Acting Medications:
When you take vitamin B6, an enzyme converts it into pyridoxal 5' phosphate, which is the active form of the vitamin in the body. Theophylline drugs, which are commonly used by individuals with asthma, are reported to inhibit the enzyme that catalyzes this conversion. Therefore, people who regularly take theophylline-containing medications are at a greater risk of developing a vitamin B6 deficiency. (8) In fact, several studies have documented that patients taking theophylline medications do have depressed levels of vitamin B6. (9) , (10) Thus, people taking theophylline medications should take additional vitamin B6.
In one study, 20 subjects with asthma were given 1000 micrograms of vitamin B12 by injection once a week for four weeks. 18 of the 20 patients experienced substantial improvement, which included reduced wheezing, improved breathing upon exertion, better sleep, and improved general condition. (11) Several other older studies also report substantial improvements in asthma sufferers receiving vitamin B12, but unfortunately, more recent studies have not been performed. (12) , (13) Vitamin B12 is also effective in reducing the incidence of bronchial asthma attacks in people who are sensitive to sulfite. (14)
As discussed earlier, the major events in an asthma attack are increased contraction of smooth muscle in the bronchial passages and hyper-reactivity of the airway lining. Magnesium helps to regulate smooth muscle contraction and relaxation. This makes magnesium an important nutrient for allowing the bronchial passages to contract and relax normally. The results of one study show a strong connection between bronchial reactivity and the amount of magnesium in the bronchial tissues. (15) In general, asthma suffers have been found to have significantly lower than normal magnesium levels. Additionally, an association has been found between low cellular magnesium levels and hyper-reactivity of the airways in people with asthma. (16)
Magnesium is also used intravenously in the treatment of children with moderate to severe asthma, resulting in significant improvement in short-term lung function. (17) Other studies report that inhaled magnesium provides a mild protective effect for asthmatic patients. (18)
Eicosapentaenoic acid (EPA) is the omega-3 fatty acid derived from alpha linolenic acid, the essential fatty acid found in fish oils and flaxseed oil. EPA helps regulate the production of prostaglandins and leukotrienes, chemicals in the body that provide anti-inflammatory effects. In one study, asthma suffers taking an 1,800 mg daily dose of of EPA had significant reductions in asthma symptoms and gains in lung capacity. (19)
The use of probiotics, supplements containing friendly intestinal bacteria such as acidophilus and bifidus, supports immune function. For many people with asthma, food intolerance can lead to inflammation. Probiotic supplements can help asthma sufferers by strengthening the gut so that less toxins are absorbed into the bloodstream. This reduces the body's load of substances that set off an attack.
Glutamine is an amino acid known to help heal and strengthen the gastrointestinal tract. Glutamine shuts down inflammation in the gut wall, making it less leaky and vulnerable to toxins and allegens. (20)
The authors of a 1994 review reported that a number of studies support the use of vitamin C in asthma and allergy. Significant results included better scores on lung function tests, reduced reactivity of the lungs when challenged by allergens and histamine, improvement in the function and mobility of white blood cells, and a decrease in respiratory infections. However, there were also negative studies, which prompted the authors to conclude that more remains to be learned about the role of vitamin C in asthma. (21) Differences in dosage levels are one big variable that could cause conflicting results.
Exercise-induced asthma is a common form of non-allergic asthma that can be beneficially affected by antioxidant nutrients. In one study, vitamin C provided a protective effect against exercise-induced asthma. (22)
Smog and other forms of air pollution can trigger asthma attacks in susceptible individuals. One study revealed that supplementation with both vitamin C and vitamin E can benefit adults with asthma who are exposed to air pollutants. (23)
Cordyceps is a unique black mushroom that extracts nutrients from and grows only on a caterpillar found in the high altitudes of Tibet and China. One of the most interesting herbs in traditional Chinese medicine, Cordyceps is used for lung and kidney problems, and as a general tonic for promoting longevity, vitality, and endurance. (24) Cordyceps is beneficial in helping individuals with decreased energy restore their capacity to function at a greater level of activity.
Cordyceps has been used in humans for centuries as a tonic for improving performance and vitality. It is believed to work by maintaining a steady supply of oxygen in tissues, thus helping the heart and lungs utilize oxygen more effectively under stress. Cordyceps may also support the immune and endocrine systems, increasing physical strength and endurance. (25) , (26)
Cordyceps has traditionally been used for its improvement in respiration and in individuals with decreased lung function, such as in asthma and bronchitis, by increasing oxygenation. (27) Cordyceps is an antioxidant that increases blood levels of the free-radical scavenging enzyme superoxide dismutase (SOD). (28) Laboratory studies point to a possible anti-blood clotting property. (29) Cordyceps may also help retard atherosclerosis (clogging of arteries by plaque) through its reported positive effect on blood fat levels. (30)
Tylophora has been used traditionally in Ayurvedic medicine for thousands of years for problems with the lungs and breathing. Tylophora is used in the nutritional support of bronchial asthma and symptoms of allergies. Numerous studies report the beneficial effects of tylophora in the treatment and management of bronchial asthma. (31) , (32) , (33) Tylophora has been reported to have short acting bronchodilator activity, but anti-asthmatic effects are believed to be more accurately explained by tylophora's reported ability to regulate the immune system. (34)
Tylophora has also been reported to have beneficial effects on the immune system, possibly stimulating immune cells that destroy allergy-causing substances while controlling antibody activity that contributes to asthmatic attacks. (35)
Coleus is a relatively new medicinal herb in the United States, although it has been extensively researched in India over the last twenty years. There have been studies to support its hypotensive (blood pressure lowering), anti-asthmatic, and anti-spasmotic (muscle-relaxing) effects. (36) , (37) Most studies have been done with the isolated active ingredient, forskolin, but current research supports that the whole plant and all its constituents may actually be more effective.
Grape seeds contain beneficial bioflavonoid-like ingredients called "proanthocyanidins" (PCOs or OPCs). Like many bioflavonoids found throughout the plant kingdom, PCOs are powerful antioxidants that gobble up free radicals. (Normal byproducts of metabolism, free radicals are highly unstable molecules that can damage tissues if not kept in check.) PCOs enhance absorption of vitamin C and work in partnership with vitamin C to reduce the body's free-radical load. (38)
Proanthocyanidins are believed to protect cell membranes by neutralizing particularly harmful free radicals called "lipid peroxides". (39) , (40) PCOs may strengthen the capillary walls and skin. PCOs have been reported to inhibit the release of inflammatory chemicals, such as histamine and prostaglandins. (41) , (42) Proanthocyanidins are claimed to neutralize many types of free radicals found in the body. (43) , (44) , (45) They may also inhibit the enzyme xanthine oxidase, an enzyme that generates free-radicals, as reported in a laboratory study. (46) PCOs have been used in allergies and asthma because of their reported ability to slow histamine release from "mast cells." (Key players in asthma and allergies, mast cells serve as sentinels in the lungs, sinuses, skin, and GI tract, which together make up our first line of defense against the outside world. In asthma and allergies the immune-related process that releases histamine stored inside mast cells is overreactive.)The PCOs of grape seed have also been reported to have PAF inhibiting ability in laboratory studies. (47)


text:
Thank you
Thank you for confirming your email address.
We hate spam as much as you do, so letting us know exactly what emails you do or do not want to receive helps us provide you with a better experience.
If you have any questions or concerns about emails you have received or about your data, preferences or privacy, you may reach out directly to us at [email protected]


text:
While nontyphoidal Salmonella (NTS) has long been recognized as a cause of self-limited gastroenteritis, it is becoming increasingly evident that multiple-antibiotic-resistant strains are also emerging as important causes of invasive bacteremia and focal infections, resulting in hospitalizations and deaths. We have constructed attenuated Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis strains that can serve as live oral vaccines and as "reagent strains" for subunit vaccine production in a safe and economical manner. Prototype attenuated vaccine strains CVD 1921 and CVD 1941, derived from the invasive wild-type strains S. Typhimurium I77 and S. Enteritidis R11, respectively, were constructed by deleting guaBA, encoding guanine biosynthesis, and clpP, encoding a master protease regulator. The clpP mutation resulted in a hyperflagellation phenotype. An additional deletion in fliD yielded reagent strains CVD 1923 and CVD 1943, respectively, which export flagellin monomers. Oral 50% lethal dose (LD50) analyses showed that the NTS vaccine strains were all highly attenuated in mice. Oral immunization with CVD 1921 or CVD 1923 protected mice against lethal challenge with wild-type S. Typhimurium I77. Immunization with CVD 1941 but not CVD 1943 protected mice against lethal infection with S. Enteritidis R11. Immune responses induced by these strains included high levels of serum IgG anti-lipopolysaccharide (LPS) and anti-flagellum antibodies, with titers increasing progressively during the immunization schedule. Since S. Typhimurium and S. Enteritidis are the most common NTS serovars associated with invasive disease, these findings can pave the way for development of a highly effective, broad-spectrum vaccine against invasive NTS.
An unexpected consequence of systematic blood and body fluid (cerebrospinal fluid, etc.) culture-based surveillance for invasive bacterial infections in sub-Saharan Africa was the discovery that in infants and toddlers in multiple geographic sites, nontyphoidal Salmonella (NTS) infections rivaled Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae infections in their frequency and severity (7, 8, 23, 32, 35, 39, 43, 46, 51, 60). Incidence rates of 200 to 350 cases of invasive NTS infections/105 infections in infants and toddlers were recorded, and it was found that the majority of invasive NTS strains were resistant to multiple clinically relevant antibiotics and had high case fatality rates (typically between 20 and 30%) (8, 23, 35, 43). While severe malarial anemia and HIV are important risk factors (21, 22), invasive NTS disease is also a major health problem in low-HIV-prevalence areas in Africa (17, 54).
Invasive NTS disease is also a significant health problem in developed countries, such as the United States. In young infants (<3 months of age), the elderly, and immunocompromised hosts (e.g., those on chemotherapy for cancer or autoimmune diseases), NTS often leads to severe clinical disease, meningitis, and death (36, 59). Among infants and the elderly (age ≥ 60 years), 25% and 47% of all reported invasive cases, respectively, end up hospitalized (36). In the United States, it is estimated that approximately 7% of NTS infections are invasive, of which ∼5% are fatal (59). NTS strains resistant to multiple antibiotics complicate the treatment of invasive NTS disease (57).
Surveys from multiple sites in sub-Saharan Africa reveal that ∼80 to 90% of NTS from cases of invasive disease are Salmonella enterica serovar Typhimurium and monophasic S. Typhimurium variants, serovars that fall into Salmonella group B, or Salmonella enterica serovar Enteritidis, a group D Salmonella serovar (7, 8, 23, 32, 35, 39, 43, 46, 60). In the United States and Europe, S. Typhimurium and S. Enteritidis account for 45 to 50% of all invasive NTS cases (1, 58). Thus, an effective NTS vaccine directed against these two serovars could provide broad protection against bacteremia due to NTS.
A live oral NTS vaccine would be logistically easy to administer in developing countries, avoiding the need for injections and disposal of needles and syringes that may be contaminated with bloodborne HIV and hepatitis viruses (53). Several attenuated S. Typhimurium strains harboring ΔaroA (29, 30), Δcrp Δcdt (63, 64), ΔphoP (18), or ΔrelA ΔspoT (45) deletions have been developed and shown to be attenuated in mice and were able to protect against a dose >104-fold above the LD50 of the wild-type parental strain. Several live attenuated S. Typhimurium vaccine strains have been licensed for use against Salmonella infections in poultry (6, 14, 25). Phase 1 clinical trials have been performed using attenuated strains of S. Typhimurium derived from classic human gastroenteritis strains. These strains have been used as live oral vaccines or as live vectors expressing foreign antigens (3, 24). However, because of reactogenicity or disappointing immunogenicity, these vaccine strains did not advance further in clinical development. As such, no attenuated NTS strains (or other types of NTS vaccines) have been licensed for use in humans.
Ideally, live Salmonella vaccine strains should harbor multiple independent attenuating mutations. In this study, we deleted guaBA, clpP, and fliD in wild-type NTS strains. The guaBA deletion, which impairs guanine synthesis, was previously shown to strongly reduce the virulence of Salmonella enterica serovar Typhi (61) and Shigella flexneri 2a strains (38). The ClpPX protease degrades the master flagellum regulator proteins FlhD/FlhC (55, 56). When either clpP or clpX is deleted, the master flagellum regulator complex FlhD/FlhC is not degraded and large amounts of flagella are overproduced. S. Typhimurium clpPX mutants are attenuated in vivo, and when given orally to mice they protect against oral challenge (44). FliD is a capping protein on the tip of the flagellar filament (27, 31), and in the absence of FliD, flagellin monomers are exported into the supernatant (26, 28). The last mutation allows us to compare the contribution of flagella when expressed as cell-attached organelles versus non-cell-associated flagellin monomers.
The aim of the current study was to determine whether invasive S. Typhimurium and S. Enteritidis strains harboring deletions in guaBA and clpPX (and fliD) would be sufficiently attenuated yet able to protect mice against a lethal oral challenge with wild-type Salmonella. A practical outcome of this work is the potential use of these attenuated strains in a bivalent live oral vaccine for protection against invasive S. Typhimurium and S. Enteritidis infection. A second potential use of these strains could be the safe and large-scale purification of NTS flagellin and polysaccharide antigens that could be used in a parenteral subunit vaccine.
Bacterial strains, plasmids, and culture conditions.The bacterial strains used in this study are shown in Table 1. Plasmids pKD46, pKD13, and pCP20 were used for chromosomal deletions (16). Plasmid pCR-Blunt II-TOPO (Invitrogen, Carlsbad, CA) was used to clone blunt-ended PCR products. Salmonella strains were maintained on animal-product-free Lennox medium (Athena Environmental Sciences, Baltimore, MD) at 37°C. NTS serovars were verified by agglutination of bacteria with O-grouping and H-typing antisera (Denka Seiken Co. Ltd., Japan). Phase switching was performed by preparing swarm agar (nutrient broth containing 0.5% agar), dropping H-i or H-2 antiserum on the surface, and then inoculating by stabbing the center of the medium. Following incubation at 37°C for 20 h, the bacteria were agglutinated with H-typing antiserum. All guaBA mutants were grown on medium containing 0.005% (wt/vol) guanine. When required, antibiotics were used at a final concentration of 50 μg/ml carbenicillin, 50 μg/ml kanamycin, or 20 μg/ml chloramphenicol. For motility tests, medium containing 1% tryptone, 0.5% NaCl, and 0.4% agar (19) was used. Chemically defined medium (50) was prepared by combining 5 ml 1 M MgSO4, 130 μl 0.1 M thiamine hydrochloride, 25 ml 20% glycerol, 5 ml trace salts solution (0.2 g d-biotin dissolved in 1 ml of 2 N NH4OH, 2.00 g CuSO4·5H2O, 0.08 g sodium iodide, 3.00 g MnSO4·H2O, 0.20 g Na2MoO4·2H2O, 0.02 g boric acid, 0.50 g CoCl2·6H2O, 0.50 g CaSO4·2H2O, 7.00 g ZnCl2, and 22.0 g FeSO4·7H2O per liter) to 1 liter base solution (13.3 g KH2PO4, 4 g [NH4]2HPO4, 1.7 g citric acid).
DNA methods.Plasmid extraction and gel purification of DNA fragments were performed using the Wizard (Promega, Madison, WI) and QIAquick gel extraction (Qiagen, Valencia, CA) kits, respectively, as directed by the manufacturer. All restriction enzymes were purchased from New England BioLabs (Ipswich, MA).
PCR amplifications were routinely performed with 1 to 2.5 U Taq DNA polymerase (Denville Scientific, Metuchen, NJ, or Genscript, Piscataway, NJ), 1× PCR buffer containing 1.5 mM MgCl2, 200 μM (each) deoxynucleoside triphosphates (dNTP), and 1 μM (each) primers in a reaction volume of 20 to 50 μl in an Eppendorf Mastercycler instrument. For PCRs using long primers (>25 bp), the amount of MgCl2 was increased as necessary. When error-free and/or blunt-end PCR products were required, VentR DNA polymerase (New England BioLabs) was used according to the manufacturer's instructions.
Construction of mutants and confirmation of phenotypes. (i) Mutagenesis.Deletion of guaBA, clpP or clpX, and fljB was achieved by lambda red-mediated mutagenesis (16) using linear DNA cassettes constructed using an overlapping PCR method based on that described by chalker et al. (9) with several modifications as described in the supplemental material. For deletion of fliD, it was imperative to preserve transcription and translation of the fliS gene, the second gene in the fliDST operon, which encodes a chaperone critical for blocking premature polymerization of FliC prior to export out of the cytoplasm (5, 62). Therefore, ΔfliD was engineered to encode a truncated nonfunctional protein comprised of 38 amino acids and a molecular mass of 4.0 kDa. The deletions were verified genotypically by PCR using primers (see Table S1 in the supplemental material) external to the deletion and by sequencing at least 500 bp both upstream and downstream of the deletion. The mutants were tested phenotypically by performing motility tests and assessing flagellin production by SDS-PAGE and Western immunoblotting.
(ii) Protein methods.Samples were prepared from cultures grown in Lennox medium without shaking at 37°C overnight. After overnight growth, the optical density at 600 nm (OD600) of each culture was determined. Bacteria were pelleted by centrifugation, and the supernatant was transferred to a fresh tube. The bacterial pellet was resuspended in 40 to 50 μl of distilled water (dH2O) and mixed with an equal volume of 4× Laemmli sample buffer (3.05% [wt/vol] SDS, 0.02% [wt/vol] bromophenol blue, 0.1 M Tris [pH 6.8], 50% [vol/vol] glycerol, 5% [vol/vol] 2-mercaptoethanol), three volumes of supernatant were mixed with 1 volume of 4× Laemmli buffer, and the samples were boiled for 5 min. Equivalent amounts of cell pellet or supernatant (based on the OD600 value) were loaded in each lane. Proteins were resolved using 4 to 20% Novex Tris-glycine gels (Invitrogen) and visualized by staining with Coomassie using GelCode blue stain reagent (Thermo Scientific [Pierce, Rockford, IL]). For Western blots, proteins were electroblotted onto a polyvinyldifluoride membrane (Bio-Rad, Hercules, CA). The detection of flagellin was carried out using the mouse monoclonal antibody 15D8 at a dilution of 1:1,000 (Bioveris, Gaithersburg, MD) and a peroxidase-labeled affinity-purified goat anti-mouse secondary antibody at a dilution of 1:10,000 (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Immunoblots were developed using an ECL Plus detection system (GE Healthcare, Piscataway, NJ), and blots were exposed to Kodak X-OMAT XAR-2 film.
(iii) Motility assays.Bacteria were stab inoculated onto motility plates using a sterile pipette tip. The plates were incubated for 17 to 20 h at 37°C, and the zone of motility (diameter in mm) was measured. The ΔguaBA mutants (CVD 1920 and CVD 1940) were tested on every plate, and the motility of each strain was expressed as a ratio relative to the ΔguaBA mutant.
LD50 analysis.All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Mice were acclimated for 7 days after arrival before starting the experiments. Ten-fold dilutions of wild-type and attenuated NTS strains were administered orally to five 7-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA). Both attenuated and wild-type Salmonella strains were grown by incubating them at 37°C in Lennox medium for 20 h without shaking. Bacteria were pelleted by centrifugation and resuspended in phosphate-buffered saline (PBS) at the appropriate concentration. Mice were infected with 200 μl of bacterial suspension using a 1.5-in. curved gavage needle with a 2.25-mm ball (Braintree Scientific, Braintree, MA). The exact number of bacteria used to infect mice was determined by viable counts. Mice were weighed and monitored daily for 28 days. Any mouse that lost >20% of its body weight (compared to weight at t = 0) or that showed signs of extreme morbidity (e.g., shallow breathing and hunched posture) was euthanized and scored as a death. The 50% lethal dose of each strain was calculated by linear regression analysis.
Shedding of vaccine strains in feces.Three 12- to 16-week-old female BALB/c mice (Charles River Laboratories) were infected by gavage with 109 CFU of the kanamycin-resistant precursors of CVD 1921 or CVD 1941 (i.e., ΔguaBA ΔclpP::Kmr) suspended in 200 μl PBS. Bacteria were grown as for the LD50 and immunization experiments, except that kanamycin was included in the medium. Three to five fecal pellets were collected from each mouse and suspended in PBS at 100 mg/ml. Fecal samples were recovered for up to 28 days after inoculation, and the number of viable Salmonella per gram of stool was determined by plating onto medium containing kanamycin. The limit of detection was 100 CFU/g feces.
Immunization and challenge of mice.Fifteen 7-week-old female BALB/c mice (Charles River Laboratories) were immunized by gavage with 109 CFU of CVD 1921, CVD 1923, CVD 1941, CVD 1943, or PBS (30 mice) on days 0, 28, and 56. Bacteria were prepared and mice inoculated as per the LD50 analysis. Blood samples from mice to obtain serum were collected on days −1, 27, 55, 70, and 83. Mice were challenged orally on day 84 with 2 × 106 CFU (100 LD50) of the parental, virulent, wild-type strain (S. Typhimurium I77 or S. Enteritidis R11). Mice were weighed and monitored daily for 33 days and euthanized exactly as in the LD50 experiments.
Measurement of antiflagellin and anti-LPS antibodies.Mice were bled before the first immunization (day −1) and then on days 27, 55, 70, and 83, and sera were stored at −20°C until tested. Serum IgG antibodies against flagella and lipopolysaccharide (LPS) were determined by enzyme-linked immunosorbent assay (ELISA). Salmonella cell-associated phase 1 flagella were isolated from CVD 1924 or CVD 1941 as previously described (52). Salmonella LPS was isolated from CVD 1921 or CVD 1941 using the Darveau and Hancock method (15). Ninety-six-well ELISA plates were coated with 100 μl of S. Typhimurium or S. Enteritidis flagella or LPS (5 μg/ml in carbonate buffer [pH 9.6]) for 3 h at 37°C and blocked overnight with 10% dried milk in PBS. The plates were washed with PBS containing 0.05% Tween 20 (PBST) (Sigma). Sera were tested in serial dilutions (in 10% milk in PBST). Antibodies bound to the immobilized antigens were detected using peroxidase-labeled goat anti-mouse IgG (KPL, Inc., Gaithersburg, MD) diluted 1:1,000 in 10% dried milk in PBST, followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (KPL). Test and control sera were run in duplicate. Titers were calculated by interpolation in a standard curve as the inverse of the dilution that produces an absorbance value of 0.2 above the blank (ELISA units). Seroconversion was defined as a 4-fold increase in the antibody titer after immunization.
Serum bactericidal activity.Overnight cultures of bacteria were diluted 1:1,000 in fresh Lennox medium and incubated with shaking at 37°C until the OD600 reached 0.2. The cultures were then diluted 1:400 in PBS to a concentration of 1 × 105 CFU/ml. Mouse sera (day 70) was diluted 1:10 in PBS. The antigen mixture was then prepared by combining 1.5 μl of diluted bacteria, 3 μl of guinea pig complement (Cedarlane Laboratories Ltd., Burlington, NC), and 25.5 μl of PBS. The entire antigen mixture was incubated with 30 μl diluted mouse sera at 37°C for 1 h. Viable counts were performed both prior to and after exposure to complement. Results were expressed as the percentage of the starting inoculum that survived exposure to mouse serum and complement. Pooled sera were tested for bactericidal activity against each strain on at least three separate occasions.
Opsonophagocytic uptake by macrophages.Mouse macrophage J774 cells were maintained using Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/liter d-glucose, 584g/liter l-glutamine, and 110 mg/liter sodium pyruvate (Gibco [Invitrogen]) supplemented with 10% [vol/vol] fetal bovine serum (FBS). Twenty-four-well plates were either seeded with 1 × 105 J774 cells/well and incubated for 2 days or seeded with 4 × 105 cells/well and incubated for 1 day. Semiconfluent J774 cell monolayers were washed once with sterile PBS, which was replaced with fresh tissue culture medium. Bacteria were prepared by performing a 1:1,000 dilution of an overnight culture in fresh Lennox medium and incubating at 37°C until the OD600 reached 0.2. The culture was diluted 1:20 in PBS. Ninety microliters of this bacterial suspension was then added to 10 μl sera (which had been heat inactivated at 56°C for 20 min) and incubated at room temperature for 20 min while rotating. A 10-μl aliquot of the cell suspension ([2 to 5] × 104 CFU) was added to a semiconfluent layer of J774 cells. The tray was then incubated at 37°C in a 5% CO2 incubator for 45 min. The cells were washed once with PBS, replaced with fresh medium containing 100 μg/ml gentamicin (which kills extracellular bacteria only), and the cells were incubated for 1 h at 37°C in humidified atmospheric air containing 5% CO2. The medium was then removed, and the cells were washed three times with PBS. Finally, the J774 cells were lysed with 500 μl of 0.5% (wt/vol) Triton X-100 (t-octylphenoxypolyethoxyethanol) and vigorous pipetting to disrupt the cells. Intracellular bacteria were enumerated by performing viable counts. Each strain was tested for opsonophagocytic uptake in duplicate wells and on at least three separate occasions. The percentage of cells taken up by J774 cells (i.e., percent opsonization) was calculated by dividing the number of bacteria that survived the gentamicin treatment by the inoculum size and multiplying by 100. For the experiments showing uptake across serovars, the results are expressed as fold uptake (the number of NTS bacteria coated with serum from immune mice which are taken up by J774 cells, divided by the number of NTS bacteria coated with PBS-serum which are taken up by J774 cells) to account for differences in basal uptake.
Statistical methods.Data were analyzed using Student's t test (two-tailed) or Fisher's exact test (two-tailed; http://www.langsrud.com/fisher.htm).
Construction of attenuated nontyphoidal Salmonella strains and their phenotypes.We chose two invasive NTS strains isolated from the blood of toddlers in Mali to use as the basis of live attenuated vaccine strains. Both S. Typhimurium I77 and S. Enteritidis R11 are antibiotic-susceptible and possess the Salmonella virulence plasmid (Table 1).
The guaBA loci of S. Typhimurium I77 and S. Enteritidis R11 were deleted using the lambda red recombinase technique. The mutants were tested phenotypically by streaking them onto chemically defined medium with or without guanine. While the respective wild-type strains, S. Typhimurium I77 and S. Enteritidis R11, grew on both media, as expected, the guaBA mutants, CVD 1920 (S. Typhimurium I77 ΔguaBA) and CVD 1940 (S. Enteritidis R11 ΔguaBA), could not grow on medium lacking guanine (data not shown).
A ΔclpP polar mutation was then introduced into the S. Typhimurium I77, S. Enteritidis R11, CVD 1920 (S. Typhimurium I77 ΔguaBA), and CVD 1940 (S. Enteritidis R11 ΔguaBA) backgrounds, resulting in I77 ΔclpP, R11 ΔclpP, CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), and CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP), respectively. Motility tests showed that, as expected, the ΔclpP mutants were 1.1- to 1.4-fold more motile than both the wild-type strains and the ΔguaBA mutants (Fig. 1; see also Fig. S1 in the supplemental material). SDS-PAGE and Western blot analyses showed that CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP), and the S. Enteritidis R11 ΔclpP mutant produce more cell-associated flagella than the parental strains (Fig. 2). Although the S. Typhimurium I77 ΔclpP mutant did not appear to hyperexpress flagella, we found that hyperflagellation could be induced by subculturing at 30°C (results not shown). Also, the SDS-PAGE and Western analyses suggested that CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) produces only FljB. Moreover, using serotyping, we found that the bacteria were able to switch phases and could be agglutinated with both H-i (phase 1) and H-2 (phase 2) antisera. We also deleted the clpX gene from CVD 1940 (S. Enteritidis R11 ΔguaBA) to produce mutant CVD 1944 (S. Enteritidis R11 ΔguaBA ΔclpX), which showed the same increased motility and flagellin production as CVD 1941, which possesses a ΔclpP polar mutation in the CVD 1940 background.
Although we anticipated that CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) and CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) would make good attenuated vaccine strains, we decided to delete one more locus to determine its effect on immunogenicity and to produce safe S. Typhimurium and S. Enteritidis reagent strains for subunit vaccine production (conjugate vaccines using flagellin monomers as the carrier protein). The fliD gene was deleted from the CVD 1920 (S. Typhimurium I77 ΔguaBA), CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1940 (S. Enteritidis R11 ΔguaBA), and CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) backgrounds, resulting in strains CVD 1922 (S. Typhimurium I77 ΔguaBA ΔfliD), CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD), CVD 1942 (S. Enteritidis R11 ΔguaBA ΔfliD), and CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD), respectively. As anticipated, the resulting mutants were no longer motile (Fig. 1; see also Fig. S1 in the supplemental material). Furthermore, SDS-PAGE and Western blot analyses show that all resulting ΔfliD mutants mainly export flagellin into the supernatant (it is no longer cell associated), and moreover, CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD) and CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) export large amounts of flagellin into the supernatant (Fig. 2). Since S. Typhimurium produces both phase 1 (encoded by fliC) and phase 2 (encoded by fljB) flagella, we also deleted fljB to produce strains that hyperexpress only phase 1 cell-associated flagella or secreted flagellin only. The fljB gene was deleted from CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) and CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD) to produce CVD 1924 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfljB) and CVD 1925 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD ΔfljB), respectively. As shown in Fig. 2, the fljB mutants produce only FliC.
Safety of attenuated NTS strains.To determine whether ΔguaBA and ΔclpP can function as independently attenuating mutations, we determined the oral 50% lethal dose (LD50) of our vaccine strains in BALB/c mice. The oral LD50 of the wild-type strains S. Typhimurium I77 and S. Enteritidis R11 was determined to be 2 × 104 CFU for both strains. First, we determined that the oral LD50 for S. Typhimurium I77 ΔclpP was >4.7 × 108 CFU. Thus, we confirmed the previous findings of Matsui et al. (44) in showing that a ΔclpP mutation greatly attenuates S. Typhimurium. Similarly, S. Enteritidis R11 ΔclpP is also highly attenuated, and the LD50 was >1.6 × 109 CFU. Finally, CVD 1920 (S. Typhimurium I77 ΔguaBA), CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD), CVD 1940 (S. Enteritidis R11 ΔguaBA), CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP), and CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) possess LD50s greater than 7.8 × 108 CFU when given orally to BALB/c mice (Table 2). These results indicate that the ΔguaBA and ΔclpP mutations are independently attenuating and that the vaccine strains CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD), CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP), and CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) each have an oral LD50 greater than 4 log10 above that of the wild-type parental strains.
Shedding of the attenuated NTS strains in mouse feces.Since our attenuated vaccine strains do not possess any antibiotic selection markers, we used antibiotic-resistant precursors to the final CVD 1921 and CVD 1941 strains (i.e., ΔguaBA ΔclpP::Kmr) to monitor shedding in feces. We inoculated BALB/c mice via oral gavage with 109 CFU of CVD 1921 Kmr (S. Typhimurium I77 ΔguaBA ΔclpP::Kmr) or CVD 1941 Kmr (S. Enteritidis R11 ΔguaBA ΔclpP::Kmr) and monitored excretion of the strains for 28 days. The number of CFU/g rapidly decreased within the first week of dosing, and CVD 1921 Kmr (S. Typhimurium I77 ΔguaBA ΔclpP::Kmr) was not detected in any of the mice at days 21 and 28 (Fig. 3). At the same time points, CVD 1941 Kmr (S. Enteritidis R11 ΔguaBA ΔclpP::Kmr) was detected in one out of three mice.
Immune responses generated by the live attenuated NTS vaccines.Mice were immunized orally with CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD), CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP), or CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) three times, 1 month apart. One mouse from each of the CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) and CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) groups died following the third immunization, possibly due to the gavage procedure. Anti-LPS and anti-FliC IgG titers increased progressively during the immunization schedule (Table 3). Mice immunized with attenuated S. Typhimurium constructs CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) and CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD) had excellent seroconversion rates after the second immunization (CVD 1921 [14/15 mice for anti-LPS and 15/15 mice for anti-FliC] and CVD 1923 [15/15 mice for both anti-LPS and anti-FliC]) and mounted robust antibody responses against both target antigens. For mice immunized with attenuated S. Enteritidis vaccine candidates, there was a trend of higher seroconversion rates after the first dose in mice that received CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) than for those immunized with CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD), which was statistically significant for anti-FliC (15/15 versus 6/15; P < 0.001, Fisher's exact test). Just prior to challenge, 15/15 mice immunized with attenuated S. Enteritidis CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) showed seroconversion for anti-LPS, and 13/15 mice showed seroconversion for anti-FliC. IgG isotype analysis was performed on sera from day 83, and the ratio of IgG2a to IgG1 titers was determined (Fig. 4). The mean IgG2a/IgG1 ratios were 0.64 and 1.23 for CVD 1921- and CVD 1923-immunized mice, respectively (P = 0.28, Student's t test, two-tailed) and 5.17 and 1.45 for CVD 1941- and CVD 1943-immunized mice, respectively (P = 0.14, Student's t test, two-tailed).
Sera from the immunized mice were also examined for their ability to directly kill NTS and to mediate opsonophagocytic uptake. Pooled sera from CVD 1921- or CVD 1923-immunized mice obtained 2 weeks after the last immunization were unable to kill S. Typhimurium I77 (Fig. 5). Sera from CVD 1941- and CVD 1943-immunized mice also showed no bactericidal activity against S. Enteritidis R11.
In contrast to the lack of bactericidal activity, the immune sera exhibited excellent opsonophagocytic activity. Individual sera from mice immunized with CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) or CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD) were able to promote significantly higher uptake of S. Typhimurium I77 by J774 cells than sera from unvaccinated mice (PBS control, Fig. 6). Likewise, individual sera from mice immunized with CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) or CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) were able to promote significantly higher uptake of S. Enteritidis R11 by J774 cells than sera from PBS control mice. In fact, the pooled immune sera generated by the CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) vaccine enabled uptake of other strains of S. Typhimurium and also two other group B serovars, Salmonella enterica serovar Stanleyville and S. enterica serovar I 4,[5],12:i:− (Fig. 7). Higher uptake of other invasive S. Enteritidis isolates and two Salmonella enterica serovar Dublin (a group D serovar) strains was also observed following opsonization with pooled sera from CVD 1941-immunized mice compared to apsonization with sera from PBS-inoculated mice. Interestingly, the pooled sera from mice immunized with a group B Salmonella vaccine were generally able to mediate uptake of group D Salmonella and vice versa (Fig. 7C). Sera from CVD 1921-immunized mice were able to opsonize and enable ∼10-fold-higher uptake of the S. Typhimurium strain S52 than sera from PBS-treated mice, whereas opsonization of this strain with sera from CVD 1941-immunized mice led to no uptake. Sera from CVD 1941-immunized mice were able to opsonize and enable uptake of the S. Enteritidis strains R11 and P149 more efficiently than sera from CVD 1921-immunized mice, though the difference was significant only for strain P149 (for R11, P = 0.057; for P149, P = 0.038 [Student's t test, two-tailed]).
Ability of attenuated NTS to immunize and protect BALB/c mice against a lethal challenge.Immunized mice were challenged with 100 LD50s (2 × 106 CFU) of S. Typhimurium I77 or S. Enteritidis R11. As shown in Fig. 8, none of the control mice receiving PBS and challenged with virulent S. Typhimurium survived at day 33, whereas 86% of the CVD 1921-immunized mice and 80% of the CVD 1923-immunized mice survived challenge out to day 33. For vaccinated mice challenged with S. Enteritidis, 13% receiving PBS survived challenge by day 33, whereas 79% vaccinated with CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) and 33% vaccinated with CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) survived out to day 33. These data clearly show that while both S. Typhimurium vaccine strains, CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) and CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD), carrying attached and nonattached flagella, respectively, are highly protective, in S. Enteritidis, only the flagellum-carrying strain, CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP), elicited high levels of homologous protection, while CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD), which exports flagellin monomers, was much less effective (Table 4).
We have constructed attenuated S. Typhimurium and S. Enteritidis strains that can serve as live attenuated vaccine strains and as reagent strains for safe purification of subunit vaccine components. We introduced ΔguaBA and ΔclpP deletions into S. Typhimurium I77 and S. Enteritidis R11, which were both isolated from the blood of toddlers in Mali. These two deletions were independently attenuating in an oral murine infection model, thereby indicating that NTS ΔguaBA ΔclpP mutants would make safe live attenuated vaccines due to an infinitesimally low likelihood that the double deletion mutant can revert back to the wild-type genotype. We also introduced ΔfliD deletions into these attenuated strains to determine whether secretion of flagellin as monomers instead of polymerization as flagella would affect the immune response and level of protection. Indeed, although CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD) produced anti-LPS and anti-FliC titers similar to those of CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) seemed to produce somewhat lower titers than CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP). The only notable significant difference between CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) and CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) was the seroconversion rate for anti-FliC after the first immunization. The lower immunogenicity of CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) was accompanied by lower protective efficacy following challenge with wild-type S. Enteritidis. On the other hand, CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP), CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD), and CVD 1941 (S. Enteritidis R11 ΔguaBA ΔclpP) exhibited excellent vaccine efficacies, i.e., 86%, 80%, and 76%, respectively (Table 4). These data suggest that NTS harboring deletions in guaBA and clpP can serve as effective live attenuated vaccines, whereas an additional deletion in fliD can render the vaccine ineffective. The discordant responses produced by S. Enteritidis ΔfliD mutants versus S. Typhimurium ΔfliD mutants may be due to differences in the role of intact flagella. In S. Enteritidis, flagella have been shown to be required for that serovar to persist in the gastrointestinal tract of rats (49), whereas fimbriae, such as SEF14, SEF17, SEF21, Pef, and Lpf, were not required to achieve infection. S. Enteritidis flagella but not fimbriae have also been shown to play an important role in pathogenesis in a day-old-chick model (2). In contrast, S. Typhimurium flagella do not appear to be a prerequisite for infection. Matsui et al. (44) observed that an S. Typhimurium ΔclpP ΔfliC ΔfljB mutant was recovered from the spleens of orally infected mice at levels similar to those of a S. Typhimurium ΔclpP isogenic mutant. Furthermore, Kodama and Matsui (37) orally immunized mice with the flagellum-defective mutants and showed protection against oral challenge with virulent S. Typhimurium. Therefore, intact flagella are not required for S. Typhimurium with a ClpPX-deficient background to persist in mice and generate a protective immune response.
Protection against Salmonella has been associated with antibody-mediated bacterial clearance by professional phagocytic cells (20). Results from the opsonophagocytic assays suggest that our live S. Typhimurium vaccine strain may be able to confer cross-protection against other invasive group B Salmonella serovars and our live S. Enteritidis vaccine strain may be able to protect against other invasive group D Salmonella serovars. However, S. Typhimurium S52 was taken up by J774 cells less efficiently following opsonization with sera from CVD 1941-immunized mice than with sera from CVD 1921-immunized mice. Similarly, S. Enteritidis P149 was taken up by J774 cells less efficiently following opsonization with sera from CVD 1921-immunized mice than with sera from CVD 1941-immunized mice, though this difference was less significant (P = 0.038 versus P < 0.001). These data suggest that an S. Typhimurium live attenuated vaccine may protect against some but not all S. Enteritidis strains and vice versa. However, further studies will be needed to determine whether this cross-protection occurs in vivo.
We have recently shown that a Salmonella enterica serovar Paratyphi A ΔguaBA ΔfliD strain, when given intranasally to BALB/c mice, produced FliC IgG antibodies that were predominantly IgG1 (as opposed to opsonophagocytosis-mediating IgG2a), whereas an S. Paratyphi A ΔguaBA mutant showed no such Th2 response bias (O. Gat, J. E. Galen, S. M. Tennant, R. Simon, W. C. Blackwelder, D. J. Silverman, M. F. Pasetti, and M. M. Levine, unpublished). These results are in agreement with those of Cunningham et al. (13), who showed that soluble FliC is Th2 polarizing whereas FliC presented as flagella attached to live Salmonella favors a Th1-type response. The difference in immune responses produced by the flagellin-secreting S. Paratyphi A and NTS strains, which herein produced IgG2a/IgG1 ratios similar to those of cell-associated Salmonella, is most likely due to the ΔclpP deletion of the attenuated NTS strains. It has previously been shown that extracellular Salmonella produces abundant FliC but in vivo, intracellular Salmonella produces only small amounts of FliC, which are insufficient for adequate T cell activation (11). However, intracellular repression of fliC no longer occurs when the ClpPX protease is absent (12). Therefore, it is conceivable that S. Typhimurium CVD 1923 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD) and S. Enteritidis CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) continuously secrete FliC both extracellularly and intracellularly, allowing for an effective stimulation of T cells and a Th1-type response. Hence, our data suggest that both the nature of the antigen (whether FliC is cell associated or secreted by Salmonella) and the location (intracellular fliC expression) are important determinants in the induction of T cell responses. These findings have ramifications for vaccine design, since they imply that an attenuated S. Typhimurium (but not S. Enteritidis, since the absence of cell-associated flagella may have crippled colonization) ΔguaBA ΔclpPX ΔfliD strain expressing a foreign antigen fused to FliC would be a potent inducer of antigen-specific T cell responses. Such a vaccine could be used sequentially with subunit vaccines (or others) known to induce antibody-biased responses in a prime-boost strategy to achieve broadened, enhanced immunity.
One of the problems with the live attenuated S. Typhimurium vaccines tested in humans to date is that they are shed in the feces for long periods. Our analysis of ΔguaBA ΔclpP NTS excretion in feces showed that the mice stopped shedding the attenuated S. Typhimurium strain by day 21 and only one out of three mice was still shedding the attenuated S. Enteritidis strain at days 21 and 28. These extended excretion kinetics may be due to coprophagy, whereby mice may have reinfected themselves with the vaccine strains. In contrast, we have found that three rhesus macaques fed 1010 CFU of CVD 1921 (S. Typhimurium I77 ΔguaBA ΔclpP) stopped shedding the bacteria by day 12 postinfection (the last Salmonella bacteria were detected on days 6, 7, and 10; A. Ault, S. M. Tennant, J. P. Gorres, M. Eckhaus, N. Sandler, S. Bao, K. Foulds, S. Livio, G. J. Nabel, M. M. Levine, and S. S. Rao unpublished results). Hindle et al. (24) found that their S. Typhimurium vaccine strain WT05 (TML ΔaroC ΔssaV) was shed for much longer in a human volunteer study. When 109 CFU were fed to three volunteers, positive stool cultures were observed until days 15, 21, and 23. Shedding of the attenuated NTS strains described in this study needs to be evaluated in human subjects, since it may be a potential safety constraint of these vaccines.
Having demonstrated the proof in principle that Salmonella strains carrying mutations in guaBA and clpPX can protect mice against lethal challenge, we will advance attenuated S. Typhimurium and S. Enteritidis candidate vaccine strains into phase 1 clinical trials. Based on the enormous burden of invasive NTS disease in infants and young children in sub-Saharan Africa, one main target of our NTS vaccines will be to prevent disease in children of <60 months of age in that continent. To protect that population, one would aim to vaccinate young infants with a bivalent live oral NTS vaccine through the Expanded Programme on Immunization at 6, 10, and 14 weeks of age, when they receive oral polio vaccine and parenteral pentavalent (diphtheria, whole-cell pertusis, and tetanus combination [DwPT], Hib, hepatitis B virus [HBV]) vaccine. Financial assistance from the GAVI Alliance and subsidized ("tiered") prices will almost certainly be required for this vaccine to be introduced widely in Africa. The oral polio vaccine experience documents that highly practical oral vaccines can prevent the systemic consequences of invasive infections. Nevertheless, some oral vaccines have exhibited some limitations in developing countries. For example, recent field trials in Africa with live oral rotavirus vaccines showed lower efficacy (4, 42) than was previously observed with these vaccines in North and South America and Europe, suggesting that in some developing country populations, oral vaccines may exhibit diminished immunogenicity or efficacy (33, 34, 48). However, the excellent immunogenicity in preschool children and the long-lived protection achieved with oral typhoid vaccine Ty21a in school-age children suggest that live oral Salmonella vaccines may constitute an exception (10, 40, 47). Acknowledging that our live oral approach may not by itself confer optimal protection in all target populations, we are taking full advantage of our reagent strains to simultaneously pursue a conjugate vaccine approach whereby O polysaccharide (OPS) is conjugated to flagellin as the carrier protein. For this approach, our engineered NTS strains CVD 1925 (S. Typhimurium I77 ΔguaBA ΔclpP ΔfliD ΔfljB) and CVD 1943 (S. Enteritidis R11 ΔguaBA ΔclpP ΔfliD) are ideal sources of OPS and flagellin since they are highly attenuated and should enable relatively straightforward, safe, large-scale purification of both components. Since these strains export nonpolymerized flagellin monomers, we can purify flagellin monomers directly from the supernatant of fermentation culture, thereby enhancing the yield and purity of purified cell-associated flagellin.
We thank Sofie Livio for assistance with agglutinations and Laura Martin at the Novartis Vaccines Institute for Global Health for helpful discussions.
Funding for this study was from the Middle Atlantic RCE Program NIAID/NIH 2 U54 AI057168 grant (M.M.L., principal investigator). R.S. was also supported by NIH T32 AI07524, Fellowship Training Program in Vaccinology (M.M.L., principal investigator).
- Received 27 April 2011.
- Returned for modification 21 May 2011.
- Accepted 20 July 2011.
- Accepted manuscript posted online 1 August 2011.
† Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.05278-11.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.