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Start by preheating the oven to 400. Then add some onion at the bottom of the pan.
I also roasted a spaghetti squash by cutting it in half, adding a little oil and roast for about 30-60 minutes depending on the size. Just season it with s, p, cayenne. You can also microwave it much faster.
Give it a try!

domain: food


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Death Ray 2000
More in Television
A daily updated summary of the week in TV.
The belatedly shown pilot to the short-lived Robert Conrad series, "A Man Called Sloane" (1979-80), has Robert Logan in the role Conrad played, a flamboyant secret agent from UNIT, a secret counter-espionage group, teamed here with lady spy Ann Turkel to retrieve a stolen device capable of destroying the world from the clutches of sinister munitions magnate and guest star Clive Revill. One of the bad guys in this film, Ji-Tu Cumbuka, as Torque, a burly giant with a mechanical steel hand, who is killed off in this pilot, unaccountably was brought back to life for the series as T. R. Sloane's deadly sidekick. Dan O'Herlihy, as The Director, also was in both the film and the series. The syndicated title is "T. R. Sloane."
Rookie police officers from a variety of backgrounds come together in the new CBS show “NYC 22.”
THE first installment of ''Malice Aforethought,'' which begins at 9 P.M. tonight on WNET-TV, Channel 13, as part of the ''Mystery!'' series, isn't a whodunit but a when-will-he-get-around-to-it. It is clear from the very first moment of the drama, which is adapted from Francis Iles's novel, that Dr. Edmund Bickleigh would be well advised to kill his wife. She is nagging and unpleasant and, even worse, reluctant to divorce him, although half the young ladies in the neighborhood are more amorously interested in Dr. Bickleigh than she. By the end of Part I, the doctor, who has been administering morphine for his wife's headaches, is beginning to think she might require something stronger.
AMONG the new crop of situation comedies, ''I'm a Big Girl Now,'' which has its premiere tonight at 8:30 on ABC, shines a little brighter than the rest. The creator and writer is Susan Harris, who performed the same functions for ABC's ''Soap.'' The setting for this confrontation between newly divorced father (Danny Thomas) and newly divorced daughter (Diana Canova) is Los Angeles. The comedy tone is strictly New York. Ben, a dentist, has lost his wife of 35 years to his business partner of 35 years. They have run off to Spain to kick up their aging heels in Barcelona. Ben moves in with daughter Diana and her own 7-year-old daughter, who is staggeringly precocious. Ever the protective father, Ben proceeds to treat Diana as a 7 year old herself. Quite reasonably, she objects. That's the basic situation. The comedy, at least in this initial episode, isn't bad.
What's On Tonight
MOST POPULAR - ARTS
- Critic’s Notebook: TV Spreads Its Blanket for Summer Viewing
- Art Review: Old Faces in New Places
- Art Review: Changing the World, Step by Step
- Henri Dutilleux, Modernist Composer, Dies at 97
- Hong Kong Welcomes the Art World
- New Guide in Venice
- A Tome to Rival the Artist Himself
- Dance Review: A Star Turn for Classicism: A Night for the Vehicle as Well as the Performers
- Ray Manzarek, 74, Keyboardist and a Founder of the Doors, Is Dead
- Music Review: Damn the Practice Sessions, Full Speed Ahead

domain: artistic


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|Monday||08:00 - 23:59|
|Tuesday||08:00 - 23:59|
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|Sunday||08:00 - 23:59|
Italy is one of the world’s most beautiful countries, while it’s a commonly held belief that Bologna is one of its most attractive cities. There you have it: the recipe for road trip heaven.
Explore the intriguing city of Bologna and the stunning surrounds of central Italy at your leisure. Simply reserve your rental car at Bologna Guglielmo Marconi Airport and away you go.
The Avis car hire desk is on the ground floor of the terminal at Bologna Guglielmo Marconi Airport, and our rental cars are parked at car park P3 on the ground floor.
With its beautiful architecture and delicious aromatic food, Bologna is a city for all the senses. The Piazza Maggiore, Bologna’s huge main square, is the city’s focal point. From here, everything is easily reached.
The city’s art and culture scene is overflowing with things to see and do. The Museum of Modern Art displays some exceptional examples of contemporary artwork, while the Basilica di San Petronio showcases exceptional architecture. This prominent attraction has stood in the central square for over 600 years. See if you can spot the second most famous leaning tower in Italy, the Tower of Asinelli, which is creeping gradually towards its twin.
Home to the oldest university in the world (founded in 1088), museums, galleries and more than 200 libraries, Bologna is a magnet for intellectuals and students from all over the world.
You can’t talk about Bologna without mentioning food. Home of spaghetti Bolognese or “tagliatelle al ragù”, classic Italian cuisine has been perfected here. Visit one of the city’s many exceptional restaurants to sample its delight. And with numerous theatres, cinemas, bars and clubs the city offers the visitor a lively night time scene. In Bologna, the entertainment starts early at 7 pm for the aperitivo, and goes on until the early hours of the morning.
The moment you collect the keys to your Avis hire car, you’re free to set off in any direction you choose without a care – just the open road ahead
While Bologna is captivating, there are many other regional attractions that deserve your attention too.
Parco del Delta del Po
Taking the A13 for 90 minutes from Bologna will transport you to Parco del Delta del Po. Covering an area of 52,000 hectares, this is no small reserve. This lush park is a place to connect with nature, much of which is unique to this region. Dense pine forests cover vast stretches of this area along the Adriatic coast. Whether you choose to go on a hike or relax with a picnic, this is a must-visit spot.
Deliciously fresh prosciutto and flavoursome aged parmesan cheese are the first things that spring to mind when one thinks about Parma, but there’s much more to this central, northern Italian destination. Beautiful works of art are on display in several galleries around the city, including the Galleria Nazionale. Head to the city centre to see astounding architecture, like the huge Palazzo della Pilotta and the Duomo. Drive along the A1 from Bologna for just over an hour to get there.
Florence is the stunning, romantic capital of Tuscany, famous for its leather goods and as a gateway to the region’s world-famous vineyards. This UNESCO World Heritage Site boasts ancient monuments, grand palaces and ornate churches, all worth visiting on your road trip. The Duomo is the city’s top attraction, offering spectacular 360-degree views from the top of its dome. Florence is just a 90-minute drive down the A1 from Bologna.
Whether you’re travelling around Bologna or further afield, an Avis hire car throws you the keys to a world of effortless exploration.
Which side of the road?
In Italy, please drive on the right side of the road.
Country driving laws
All speed signs will be in km/h.
For a standard Avis rental vehicle with no trailers:
Unless indicated by road signs.
Be aware of changes to speed limits displayed on road signs due to adverse weather conditions.
Child safety / Seatbelt laws
Please note - It is the child’s parent / guardian or vehicle renter’s responsibility to fit the child seat.
This road rules information is for provided for general guidance only. We endeavour to keep the information up to date and accurate, but any reliance you place on this information is at your own risk.

domain: geography


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In my personal hierarchy of "music games that sucked up way more of my free time than is reasonable by any conceivable definition," Rock Band and Guitar Hero perhaps unsurprisingly sit atop the heap. However, surprisingly not far behind them is AudioSurf, the independently developed PC music game that let you "ride" your own music. Just load up any mp3 file from your hard drive, and the game would generate a dynamic level to "surf," creating collectible objects and obstacles around specific beats and notes within the music.
It was a sincerely amazing game, so you can imagine my excitement when I learned just now of AudioSurf Air, a sequel currently in the works courtesy of creator Dylan Fitterer. The just-launched website for the game only shows a few teasing screenshots, but also has the benefit of a beta sign-up form for those who might want to try the game ahead of release.
The beta currently doesn't have a launch date, but signing up will get you considered for when it does launch. Presumably it won't be too far off, as the site lists a 2012 release date for the final game.
Okay, so this is maybe a slightly self-indulgent post given my absurd love of the original, but come on, I can't possibly be the only one stoked about this, can I?


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Moonfall - a film about total catastrophe about the fall of the Moon to Earth
You almost always know what to expect from a Roland Emmerich film: namely, the fact that with it a good part of the planet will be destroyed.
Photo Credits: Promo
You almost always know what to expect from a Roland Emmerich film: namely, the fact that with it a good part of the planet will be destroyed. The director of films like 2012 and The Day After Tomorrow now brings this to its logical extreme with the film Moonfall. A film about the falling moon….
According to the synopsis, the action of the film takes place at a time when "a mysterious force throws the Moon out of orbit around the Earth and sends it into collision with life as we know it."
It stars Halle Berry as the executive director of NASA who seems to know how to stop it, but the only people who trust her are former astronaut (Patrick Wilson) and conspiracy theorist (John Bradley). "These incredible heroes will embark on an impossible last mission into space, leaving behind all they love, only to discover that our Moon is not what we think it is."
Indeed, Moonfall sounds like something that would have happened if you had crushed a Hollywood disaster movie with Destiny lore - that is to say, it sounds like something very fun and tense. It is expected to hit theaters on February 4, 2022.
By: Amber V. - Gossip Whispers


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IHG Reports 2014 Half Year Results
August 5, 2014 10:51am
Richard Solomons, Chief Executive of InterContinental Hotels Group PLC, said:
"We have achieved a strong first half performance, with our preferred brands continuing to drive good momentum through the second quarter. With underlying operating profit2 up 6% and solid net system growth, our long-term winning strategy is delivering results. This has given us the confidence to increase the interim dividend by 9%.
We have had our best half for signings4 in six years, underpinning our future growth prospects and demonstrating owners' preference for our brands. Openings included the first two EVEN Hotels in the US, a major milestone for this new brand, which satisfies a previously unmet guest need in the wellness segment.
We remain committed to reducing the asset intensity of the business, completing two asset disposals in the half, and good progress is being made with the strategic review of our remaining owned hotels. During the half we completed our $500m share buyback programme and in July we paid a $750 million special dividend, continuing our long track record of returning funds to shareholders.
Looking ahead, whilst several of our key markets continue to experience some political or economic uncertainty, we are encouraged by current trading trends."
Driving Market Share
o Underlying profitability reflects adjustments for a net year on year operating profit impact of $(45)m, comprising: $(15)m from disposal of owned hotels; $(33)m from significant liquidated damages receipts, and $3m from managed leases.
o Americas 6.7% (US 6.6%); Europe 4.9%; AMEA 3.7%; Greater China 4.3%.
o Q2 comparable RevPAR up 5.7%: Americas 6.7% (US 6.7%); Europe 4.1%; AMEA 3.6%; Greater China 4.6%.
o 17k rooms (109 hotels) opened, led by 9k rooms in the Americas and 4k rooms in Greater China. 11k rooms removed in-line with our on-going commitment to quality.
o Pipeline of 187k rooms (1,175 hotels), over 45% under construction and over 50% in developing markets.
o Signings of 30k rooms (208 hotels), our best H1 for underlying4 signings since 2008, supported by continued improvements in the US financing environment.
o With 5% share of global industry supply, and 13% share of the active industry pipeline, we remain well positioned for sustainable high quality growth.
o First two hotels opened for our new EVEN Hotels brand in June, with an excellent initial guest response.
o Holiday Inn and Crowne Plaza first half outperformance vs their US industry segments5.
o Holiday Inn ranked "Highest in Guest Satisfaction Among Mid-scale Full Service Hotel Chains" by J.D. Power and Associates for 4th year running.
o Strong growth in mobile bookings: up 47% year on year.
o Strategic relationship with Amadeus formed as we continue to drive innovative solutions to enhance guest experiences across the Guest Journey.
o Driven by cost efficiencies and scale benefits, with some impact from favourable cost phasing.
1 All figures are before exceptional items unless otherwise noted. Refer appendices for further financial information and definitions.
2 At constant exchange rates (CER) and excluding owned asset disposals, significant liquidated damages, and results from managed lease hotels. Underlying adjusted EPS is based on Underlying EBIT and uses effective tax rate and interest as reported at actual exchange rates.
Including exceptional items.
4 Adjusted for rooms on US Army bases: 2014: 2k signed, 2013: 4k signed and opened.
5 On a Total RevPAR basis.
Americas - Strong RevPAR growth and signings pace
Comparable RevPAR increased 6.7%, with 3.1% rate growth, and second quarter RevPAR also increased 6.7%. US comparable RevPAR was up 6.6% in the first half and 6.7% in the second quarter.
Reported revenue decreased 5% (CER (5)%) to $435m and reported operating profit decreased 5% (CER (5)%) to $268m, but on an underlying basis1, revenue increased 9% and operating profit increased 7%. This was driven primarily by our franchise business where royalties were up 7%. Underlying1 owned and leased hotel profits increased 80%, driven by 6.7% RevPAR growth at InterContinental Boston, and 30.5% RevPAR growth at Holiday Inn Aruba, which is benefiting from its recent refurbishment. Recent changes to the Venezuelan exchange rate had an unfavourable impact on managed operating profit of approximately $2m, with a further $2m impact expected in the second half.
We opened 9k rooms (74 hotels) in the half, including the first two properties for our new EVEN Hotels brand. Signings of 19k rooms (158 hotels) are up 25% year on year2, as the hotel debt financing environment continues to improve, and included over 100 hotels (12k rooms) for the Holiday Inn brand family. Maintaining the quality of our system by removing hotels that no longer meet the requirements of our brands remains a key focus, and we removed 7k rooms (54 hotels) in the first half. Excluding removals, gross rooms growth was 4%.
The refurbishment of InterContinental New York Barclay is due to commence in the third quarter, with the hotel closing for up to 18 months. IHG's share of the second half loss associated with the hotel joint venture is still expected to be around $5m, which will be reflected in Americas managed results.
1 At CER & excluding owned asset disposals, significant liquidated damages and results from managed lease hotels. Year on year operating profit impact of $(31)m comprising (a) results from owned assets disposals of $(7)m ( H1'14: $(1)m; H1'13: $6m) (b) significant liquidated damages receipts of $(24)m (H1'14: $7m (franchised) of which $4m was previously announced; H1'13: $31m (managed)) and (c) results from managed lease hotels of $nil (H1'14: $1m, H1'13: $1m). Revenue impact set out in appendix 5
Excluding rooms on US Army bases: 2014: 2k signed, 2013: 4k signed and opened.
Europe - Strong trading performance in key markets
Comparable RevPAR increased 4.9%, with second quarter RevPAR up 4.1%. First half trading was particularly strong in the UK, up 8.7%, with high single digit growth in both London and the provinces, while Germany delivered another solid performance with RevPAR up 3.1%.
Reported revenue of $182m was down 12% (CER 16%) and reported operating profit of $38m decreased 28% (CER 32%), but on an underlying basis1, revenue was down 2% and operating profit down 3%. This reflects good operating profit growth in the managed and franchised business driven by mid-single digit RevPAR growth, offset by a $7m operating profit decline at our only remaining owned hotel in this region, InterContinental Paris - Le Grand (with $7m associated revenue decline). As previously guided, this was primarily due to the refurbishment of the historic Salon Opera ballroom and c.15% of the guest rooms at the hotel, with an additional small negative impact from the absence of the biannual Paris air show in 2014. No further impact is expected from the refurbishment for the full year.
We opened 3k rooms (18 hotels) in the half, including three Hotel Indigo hotels in the prime city locations of Rome, Madrid and St Petersburg. We signed 2k rooms (14 hotels) including two Holiday Inn hotels in Germany and three Holiday Inn Express hotels in the UK.
1 At CER & excluding owned asset disposals, significant liquidated damages and results from managed lease hotels. Year on year operating profit impact of $(16)m comprises (a) results from owned assets disposals of $(8)m ( H1'14: $nil; H1'13: $8m) (b) significant liquidated damages receipts of $(9)m (H1'14: $nil; H1'13: $9m (franchised)) and (c) results from managed lease hotels of $1m (H1'14: $nil, H1'13: $(1)m).
AMEA - Rate driven RevPAR growth and increasing contribution from developing markets
Comparable RevPAR increased 3.7% driven primarily by rate growth, with second quarter RevPAR up 3.6%. Excluding Thailand and Egypt where there has been ongoing political unrest, first half RevPAR increased 5.4%. Performance was led by Japan up 8.8% and South East Asia which, excluding Thailand, was up 7.2%. Australia and the Middle East continue to perform solidly with RevPAR growth of 6.0% and 4.1% respectively.
Total RevPAR grew 1.7%, reflecting an increasing mix of new rooms opening in lower RevPAR developing markets.
Reported revenue increased 15% (CER 19%) to $117m and operating profit decreased 7% (CER (7%)) to $38m. However, on an underlying basis1 revenue was flat, and operating profit decreased 12%. This reflects solid underlying growth in our managed business offset by a $2m increased investment to support future growth, a $3m negative impact from certain small one-off items, and $1m lower fees from our hotels in Thailand. The continuing political unrest in Thailand is expected to have a $2m impact on managed operating profit in the second half of the year. In addition, the managed hotel refurbishment programmes scheduled to take place in 2014, as previously disclosed, had no material impact on fees in the half, but are expected to have a $2m negative impact on fees in the second half of the year.
We opened 2k rooms (8 hotels) in the half, including the 442 room Holiday Inn Express Clarke Quay in Singapore and two Holiday Inn hotels in Japan. We signed 2k rooms (8 hotels) in the first half, including two new hotels in Abu Dhabi, which will mark the entry of a second InterContinental and second Holiday Inn property into the UAE capital.
1 At CER and excluding results from managed lease hotels. Year on year operating profit impact of $2m comprises results from managed lease hotels (H1'14: $2m, H1'13: $nil).
Greater China - Continued industry outperformance
Comparable RevPAR increased 4.3% driven by occupancy growth, with second quarter RevPAR up 4.6%. This performance was significantly ahead of the industry, which continues to experience a number of challenges including slower macro-economic conditions and austerity measures. Our industry outperformance reflects the strength of IHG's brands in the region and the leading position we have built up over 30 years of operating in the Chinese market.
Total RevPAR for the region decreased 1.4% reflecting an increasing mix of new rooms opening in lower RevPAR developing markets as we increase our distribution across the country.
Reported (and CER) revenue and operating profit were in line with last year at $112m and $36m respectively. This reflects good growth in the managed business, where 16% net rooms growth drove strong incremental fees despite the total RevPAR declines. In addition, the on-going industry austerity measures have continued to impact our food and beverage revenues in the region, up 5% compared to rooms revenues up 9%. InterContinental Hong Kong, our only owned hotel in the region, reported a $3m decrease in revenue to $66m and a $3m decrease in operating profit to $19m due to the continuing impact from the significant redevelopment of the area adjacent to the hotel; this is expected to continue into the second half.
We opened 4k rooms (9 hotels) in the half, including five Crowne Plaza hotels, taking our system size for the brand in the region to 25k rooms. We signed 7k rooms (28 hotels) taking the pipeline to 55k rooms and reflecting the confidence owners have in IHG and the compelling long term growth opportunity for this region.
Sources and uses of cash
o Strategic review of opportunities for further asset disposals is progressing well.
o Disposals of InterContinental Mark Hopkins San Francisco and an 80% interest in InterContinental New York Barclay completed with net cash proceeds of $346m.
o $13m proceeds received from recycled investments in the first half.
o $98m capital expenditure in the first half comprised: $52m maintenance capex and key money; $27m recyclable investments; and $19m system funded capital investments.
o Gross capex guidance remains unchanged at up to $350m per annum into the medium term.
o $763m1 returned to shareholders in July via $2.93 per share special dividend with 12 for 13 share consolidation.$500m share buyback programme completed, including 3.4m shares repurchased for $110m in 2014.
o $763m1 returned to shareholders in July via $2.93 per share special dividend with 12 for 13 share consolidation.
$500m share buyback programme completed, including 3.4m shares repurchased for $110m in 2014.
o Ordinary interim dividend up 9% to 25¢ reflects confidence in IHG's future prospects and our cash generative business model.
1 The difference from announced $750m is due to exchange rate movements.
Interest, tax, net debt, and exceptional items
Download the Appendices for the Interim Results to 30 June 2014 PDF (1.02Mb)
q2 2014 results
InterContinental® Hotels & Resorts, Hotel Indigo®, Crowne Plaza® Hotels & Resorts, Holiday Inn® Hotels & Resorts, Holiday Inn Express®, Staybridge Suites®, Candlewood Suites®, EVEN™ Hotels and HUALUXE® Hotels and
IHG manages IHG® Rewards Club, the world’s first and largest hotel loyalty programme with over 80 million members worldwide. The programme was relaunched in July 2013, offering enhanced benefits for members including free internet across all hotels, globally.
IHG franchises, leases, manages or owns over 4,700 hotels and 693,000 guest
rooms in nearly 100 countries, with almost 1,200 hotels in its development pipeline.
InterContinental Hotels Group PLC is the Group’s holding company and is incorporated in Great Britain and registered in England and Wales.
Visit www.ihg.com for hotel information and reservations.
Contact: Investor Relations (David Kellett; Isabel Green)
+44 (0)1895 512176 +44 (0)7808 098724
Contact: for media: (Yasmin Diamond; Zoe Bird)
+44 (0)1895 512008 +44 (0)7736 746167
IHG and Precinct Propteries Sign Agreement for 244-Room InterContinental Auckland Set to Open in 2022
IHG and Shimao Group Plan Q4 2018 Opening of InterContinental Shanghai Wonderland
Lotus Trans Travel and IHG Sign Portfolio Deal for Four Holiday Inn Hotels in India
Martyn Barnes to Bring IHG's New voco™ Brand to Australia's Yarra Valley in 2021
Marriott, Hilton, and IHG Top Franchise Companies and Brands in U.S. Construction Pipeline
IHG Opens First Hotel Indigo in Mississippi, The Hotel Indigo®-Hattiesburg
IHG Signs With Mulpha Australia Limited for Management and Rebranding of 166-Room Hayman Island, by InterContinental Opening in 2019
IHG Appoints Angelo Zuccala as General Manager for InterContinental Sofia
IHG and Ninety Four Feet Partner to Debut New Zealand's First Hotel Idigo With 2021 Opening of Hotel Indigo Auckland
IHG and TSUKADA GLOBAL HOLDINGS to Debut Kimpton Brand in Japan With 2020 Opening of Kimpton Tokyo Shinjuku
IHG Signs With Pearl Grand Tower Hotel for InterContinental® Colombo Slated for 2019 Opening in Sri Lanka
IHG Completes Acquisition of 51% Stake in Regent Hotels & Resorts for $39 Million
IHG Signs With Hotel Marina Agra for Holiday Inn Agra MG Road in India
Kimpton Saint George Opens in Toronto With General Manager Bertrand Mangeot at the Helm
IHG Opens 459-Key InterContinental® Phu Quoc Long Beach Resort in Vietnam
U.S. Project-Based Business Travel Spend Topped $45 Billion in 2017
IHG Launches New Upscale Brand, voco, With a Signing in Australia
Inuyama, in Japan’s Aichi Prefecture, to Debut Hotel Indigo, Its First Internationally-Branded Hotel
IHG Partners With SDB Developers to Expand Presence in Kolkata with Signing of Holiday Inn Resort Kolkata NH6
DUR Hospitality and IHG Open the 304-Room Crowne Plaza Riyadh Palace
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domain: finance


text: Welcome to Queen of Arts Studio, where you can receive the full artist experience. Queen of Arts Studio is uniquely built around equal opportunity and an fun family atmosphere. You do not have to be an artist to enjoy! We create memorable experiences through the arts. There is fun and entertainment for all ages.

domain: entertainment


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The stage is set for the next big Apple-Google throwdown: indoor location tracking. Apple has just been granted yet another seemingly obvious patent, this time for tracking a device’s location using WiFi access points and a location database.
Here’s how Apple’s newly-patented system works. Devices with GPS sensors connect to access points. They then relay pertinent information (latitude, longitude, SSID, signal strength, other nearby SSIDS) back to Apple’s servers which tuck that data away for later use. When a device without a GPS connects later on, its location can be approximated using the info stored in the database.
This particular patent was filed mid-way through 2010. Interestingly enough, that was right around the same time Apple pushed out iOS 4 and started silently siphoning off location data from everyone with an iPhone or 3G iPad. Early in 2011, the situation blew up and Apple found themselves on the receiving end of several lawsuits including one in Korea and one here in the United States.
Apple offered up a fix in the Spring of 2011, though they’re still gathering location data by default — they’re being more selective about what data they phone home with now.
Will this patent give Apple an edge in the war for indoor location supremacy? No, but it’s one more nice addition to the cache following their purchase of WiFiSLAM in 2013. They’re likely loading up for another cold war with Google, who themselves have around 100 indoor location-related patents. Other big tech players — including Microsoft, Nokia, and Cisco — have their fair share of indoor location IP, too.
Things could get very ugly in the near future… unless, of course, these companies all take a much different approaches to indoor location patents than they have to things like mobile operating systems.


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I just installed GeoNode and really love the manner in which it helps me arrange the contents related to the map but I am not sure about adding OpenLayers to it.
Well here is where I am faltering,
I am not sure where the OpenLayers code goes? I felt that I could use the code to customize GeoNode's look and feel and make use of the customization features on the Web Page, but to publish the Map you receive a embed link which only gives you the Map and not the other features.
If we have to use OpenLayers separately, like in the HTML, I am not sure, How to run that static html file, since the localhost is being used by the GeoNode.

domain: design


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Your dog may be uncomfortable with your husband because she was not exposed to many men as a puppy, or maybe she wasn’t exposed to men who look like your husband. … Your dog may be picking up on other, more subtle factors in her interactions with your husband, such as the way he approaches her.
What does it mean when your dog doesn’t like your boyfriend?
There may be various reasons that your dog is guarding you. Some dogs feel a need to protect and others may have had a past of bad treatment and simply may not trust men because of this. … Sometimes it can be because your boyfriend just doesn’t know how to interact with your dog the right way.
Why does my dog only dislike one person?
Dogs were found to prioritize the scent of humans over other scents. Typically when a dog sniffs a person they are uncomfortable with, it’s due to the pheromones that person is giving off. To the dog, those pheromones may signal a threat or an alarm telling the dog to beware.
Do dogs get jealous of owners partner?
According to experts, many breeds of dogs are prone to jealousy in response to their owners’ attention being diverted. Their naturally affectionate personalities can prevent them from stepping back, which can often lead to your dog displaying jealous tendencies towards you or your partner.
How do I get my dog to like my partner?
Scents and sensibility
Give your pet something with your partner’s scent on it before introducing the two. This will help your dog or cat feel more familiar with your partner when they meet. Using a pillowcase or towel works well, but skip shoes and socks, as your pet might chew on or take possession of them.
Why does my dog only like my partner?
In some cases, both partners in a couple walk and feed the dog equally, yet the dog still prefers one partner over the other. Often, this is simply a case of access, or lack of it. If one person spends more time at home with the dog, naturally the dog will want to see and spend time with the other partner, too.
Can dogs tell who your partner is?
Forget turning to your best friends when you’re unsure about the new guy you’re dating—the best judge of his character could actually be your dog. A new Japanese study shows that dogs can sense when humans mistreat their masters.
Can a dog sense a bad person?
Studies have shown that dogs take note of people who are unkind in any way to their owner and will then hold a grudge against them. … Other studies have shown that dogs can smell chemical changes in people’s pheromones that indicate they have bad intentions, therefore signalling the dog that they’re a ‘bad’ person.
Can dogs sense when you dont like them?
Trust a Dog’s Instincts–There’s a Reason They Don’t Like Someone or Some Place. … Because of their heightened sense of smell, dogs can detect things like alcohol, drugs, blood, sweat or explosives when humans can’t.
Why is my dog friendly with some dogs and not others?
Aside from their heightened sense of smell, another reason why dogs may seem to dislike other dogs instantly can be from historical experience. … Conversely, dogs may take a liking to other dogs that remind them of a certain well-liked canine friend. Your dog’s behavior towards other dogs may also be about you.
Why does my dog not like my girlfriend?
Why does my dog not like my girlfriend? Dogs do have a good sense for attitudes, but there might be a few other things to consider: Have you been giving less affection, attention, quality time to your dog without realizing, could be a slight seperation anxiety. … She might be anxious or protective of you.
Why does my dog get between me and my girlfriend?
They Are Seeking Attention
Some dogs are attention seekers—plain and simple. They notice that something interesting is going on, and they want to be part of it. If this is the case, your dog might try to join in by licking you and wagging their tail or barking. Rover may simply be looking to get his dose of attention.
What is the most jealous dog breed?
What is the most jealous dog breed? French Bulldog. If you have ever owned a Frenchie before, you know this dog clinches the title of the most jealous dog. …
How do I get my dog to like my new boyfriend?
For many dogs, the way to their heart appears to lie in their stomach. If your pooch is treat responsive and loves special treats or toys, this is a great tactic for bonding your boyfriend and pup. A can of special treats close to the door, or even in the most cooperative boyfriend’s car, is a great idea.
How do I introduce my dog to my boyfriend?
How to Introduce Your Dog to Your Partner
- Borrow an item of your partner’s clothes (in a non-creepy manner) Dogs use their nose as a means to shake hands, explore new places, and judge character. …
- Introduce them on neutral ground. …
- Tell your partner to make first contact slowly. …
- Bring your dog on your dates.
Can a dog come between a relationship?
While some dogs will leave you much-needed space and time with your partner, others simply don’t accept the fact that they are not the center of attention all the time. Some dogs can even get jealous of their owner being all cuddly with their partner to such an extent that they always choose to sit between the couple.

domain: pet


IONA-MCGREGOR, Fla. — On Tuesday, Piccadilly Posh Consignment LLC. owner Tara Golub said there has been little to get excited about during the four short months at her store’s location.
“You know I was so excited about it, a lot of people found out that I bought it and it started to really take off and then ya know Ian,” said Golub.
Golub said her store along with many others experienced storm damage as Hurricane Ian made its way through southwest Florida.
“The whole roof had caved in, in the back and everything was just, kind of muddy,” said Golub.
Muddy, a word Golub said she hates to use when it comes to describing a consignment store full of items she is trying to help others sell.
On Tuesday, Golub pointed out the marks on several pieces of furniture that were left by the storm surge that flooded her building.
Golub said she can no longer sell any of these items.
“So this stuff I am going to make for hurricane victims,” said Golub.
A gift Golub said is for the Iona-McGregor community that has supported her while also dealing with their own struggles caused by Ian.
“They keep calling me and telling me that, ya know, it's going to be okay my family ya know everybody has got good attitudes,” said Golub.
On Tuesday, Golub said she will start accepting items for resale starting on Saturday, November 5, 2022, and will be ready for in-store sales the following week.
“So it looks like we are about a week out from doing this, so it's hopeful,” said Golub.
Click here to connect with Piccadilly Posh Consignment LLC.


Men step core a Tata Motors display area on the outskirts of Agartala, Tripura on 7 Nov 2012 [File Photo].Reuters
Allocation prices of Bharat’s leading machine creator Tata Motors wine harshly on Weekday, as the society is reportedly execution the manufacturing of a non-Nano heap fully move at Sanand mill in Gujerat.
Tata Motors shares stirred an intra-day tall of Rs 338.75, in the past pare whatsoever gains to business at Rs 337.95, up 3.87%. Its shares possess fallen by way of practically 45% in the defunct sextet months.
The companionship is situate to set in motion its earliest non-Nano short auto close to Dec that yr, Province Archetype report.
“From the method facets are at the wink of an eye, it seems the presence drive be masterly to bump into rendezvous with the Dec rollout deadline,” whispered a Tata Motors vender.
Regulations christian name ‘Kite’, the brand-new motor car is nature stacked on a “entirely unusual stage.” Kite drive be a hatchback emotionless next to a 1,200-cc mechanism.
“It is seen bounteous a imaginative contract of entity to the Sanand vine, which has archaic struggling to persevere in in good volume employment levels,” the write-up understood.
Tata Motors is too in the activity of underdeveloped other original motor vehicle supported on Nano rostrum, code-named ‘Pelican’. Pelican is according to be fit purchasable next to incoming period, which disposition be a 900-1,000-cc hatchback.
Tata Motors report virtually 49% tear in its earnings in the primary ninety days of contemporaneous 1 period (2015-16) fitting to declining retailing in the life’s largest motor market-place, Dishware.
A downturn in garage sale is large go in front past slow commercial enlargement in its opener supermarket Ware and fading consumer sentiments in Bharat. Chinaware is the biggest bazaar in the service of Tata Motors’ secondary Panther Solid ground Traveller (JLR), accountancy as a service to 20% of its transaction.
But numerous analysts are at present rotary bullish atop of the store, winning into statement sale-priced valuations and anticipated gross income evolution from Service.


text: The enormous problem of workplace injuries and deaths continues to beset countries. Reflexive OHS regulation often places primary responsibility on employers’ management of OHS in organizations. This paper seeks to ascertain how OHS management at the organizational level has been treated in the research literature. A review of leading journals (13 in management, 6 in HRM) from 1994 to 2005 showed OHS management to be largely missing as the subject or field of study. Naturally, the OHS literature was more fruitful: 5 main categories were identified. However, there was little in the way nuanced explanation of OHS management at the organizational level. The paper concludes by arguing for an in-depth, contextually embedded, multi-narrative examination of successful, exemplary OHS management.

domain: library


December 29, 2012
Looking Back At Space Industry In 2012 Means Looking Ahead
Lee Rannals for redOrbit.com — Your Universe Online
2012 has been an historical year in the space industry, and the beauty of this past year is it is just paving the way for the best that is yet to come.During the past year, we have seen NASA encounter the Red Planet with their latest piece of technology, and have seen great strides made by the commercial space industry.
Last year, we ended the year on a bit of a sour note, but with hopeful dreams the United States would soon be back at its former glory. We saw a space shuttle program ended after 30 years, and a hopeful future, but no tangible evidence the future was as bright as one would hope.
This year, however, we have seen that tangible future come into play, along with the space agency with the household name making a full comeback in the media spotlight.
During 2012, NASA accomplished a feat no other space agency has ever accomplished with its successful landing of the Mars Curiosity mission. I witnessed NASA engineers at Jet Propulsion Laboratory complete a task so unreal I had to pinch myself to ensure it wasn't in a science fiction movie playing out before my hallucinogenic eyes.
NASA took the most complex rover ever dreamed-up and sent it over 300 million miles to our neighboring planet on a mission to look into the past for the presence of extraterrestrial life. In order to accomplish this mission, Curiosity had to be equipped with such complicated scientific instruments a new way of landing a rover on Mars had to be invented in order to ensure the safety of the multi-million dollar equipment.
JPL engineers went with a wild-hair idea to go ahead and send Curiosity down towards the Martian surface via a "Sky Crane" technique, which was essentially a hovering elevator capable of helping to bring the rover from a 20,000 mph velocity to nearly 0 in a matter of seven minutes. The adventure became known as the "seven minutes of terror."
Not only did NASA find success in its crazy idea, but also the space agency saw itself spreading inspiration across America for different types of entrepreneurship in the space industry.
Since the space shuttle retirement, U.S. commercial companies have been working on ideas to try and relieve America's dependence on Russia for rides to the International Space Station, as well as furthering ourselves as a dominant member of the space community.
In doing that, the U.S. has accomplished a task no other country has ever done, by having SpaceX, during a resupply mission, become the first private company to ever dock with the International Space Station.
SpaceX created its Dragon capsule with a couple of purposes in mind. The first was being able to help resupply astronauts on the ISS, and the second being the ability to send astronauts to orbit and fulfill a void left by the cancellation space shuttle program.
The Elon Musk founded company saw its first steps become reality earlier this year as its Dragon capsule docked with the ISS during the first of 12 NASA contracted resupply missions to the orbiting laboratory.
For the next few years, not only can we wait for NASA to lay out some ambitious plans to reach an asteroid and maybe Mars with human feet, but we will also be seeing U.S. companies become more dominant players in the space industry. We can look forward to new endeavors from visionaries such as Musk, who wish to eventually establish a thriving space tourism industry. But until that day comes, we can always remember 2012 as a milestone; not only for human exploration, but as a stepping stone into the future of the U.S. space industry.


The constant involvement BBNaija housemates in all events has sparked reactions from different people. This complain is coming after the involvement of BBNaija housemates in the Black Panther: Wakanda Forever premiered in Africa on Sunday, November 6, at Filmhouse Cinemas IMAX, Lagos.
Many said they were very excited about the event because some of the actors in the movie were attending and they liked the idea of people from the same industry in different countries coming together to celebrate a blockbuster.
But while a few Nollywood stakeholders and actors attended, they realised a large number of BBNaija alumni in attendance. The involvement of the BBNaija people stile the whole idea of the event at the end of the day.
All the pictures shared online showed only them like it was a Bbnaija event, they outnumbered the actual industry stakeholders. In an event that should’ve hosted legendary Nollywood actors, movie critics, directors and producers, instead, we saw ex-housemates of the reality show fill up the red carpets. But why?
Most entertainment events in Nigeria have become BBN affairs. It seems BBNaija stars are at the top of every (Public Relations) PR list these days. When Fenty launched in May, the PR agency thought it would be wise to invite them instead of actual beauty influencers who would have given target audiences more in-depth content about the launch and products.
The problem now is that when former BBN housemates attend high profile events, they simply become fashion shows. The whole idea will be uttered. We barely hear much about the actual movies, products or anything being promoted during or after the events.
At movie premieres, all we see are the same set of people with their sparkly and sometimes outrageous outfits. There are industry professionals, veterans and relevant influencers who would do better jobs at covering important talking points, so why not invite more of them?
I understand why PR agencies invite BBN influencers. They get people talking and create buzz around their events. People are still talking about what Hermes, Elo, Prince and the rest wore to the Black Panther Premiere, even though days have passed. People like Tacha trend for months on end because her fans keep talking about her.
When events revolve around BBN stars, it’s difficult not to think that these events are being diluted. Even the publicity they bring focuses on their personal brands than on what said events are about. All these reality TV stars do is dress up, create content around their outfits, show up and post pictures with uninspiring captions


Sevilla 3-2 Real Madrid: LaLiga Santander 2017-18
Despite scoring twice late on, Zidane's B team fell apart again as Sevilla earned victory with strikes by Ben Yedder and Layún, and a Ramos own goal. Jamaica vs USA: Gold Cup live
Sevilla 3-2 Real Madrd: match report
Real Madrid showed up to the Ramon Sanchez Pizjuan with their second unit and they performed like one as Sevilla outplayed them for most of the game on Wednesday night.
There were chances for Jesus Vallejo, Dani Ceballos and Theo Hernandez along with a mixture of young and old all over the field for Real Madrid. It hardly worked though as Sevilla took the lead after 26 minutes after some very generous defending from Vallejo and Sergio Ramos.
It didn't get much better from there for Real Madrid as Miguel Layun buried Sevilla's second on the stroke of half-time. It was more questionable defending from the visitors.
Sergio Ramos missed a penalty after Asensio was fouled in the area. He rattled the crossbar and would score an own-goal in the 84th minute from Gabriel Mercado's cross from the right.
Borja Mayoral headed home a consolation with three minutes to go and Ramos scored a penalty, his second attempt, when Mercado pushed Theo Hernandez in the chest right in front of the referee.
Ramos became the dubious owner of a rare record when he sent Soria the wrong way as he became the first player to miss a penalty, score an own goal and score a penalty in a game in the Spain's top flight.
Zidane won't be too upset given there's little to play for and says he won't reproach the players for the effort in the second half. There is that worry, however, that this form will creep into their form in Europe and that's when it becomes problematic.
Sevilla vs Real Madrid live stream online: minute-by-minute
Sevilla vs Real Madrid live stream online: preview
Sevilla and Real Madrid play at the Ramon Sanchez Pizjuan on Wednesday night in a game that was re-fixed for the Copa del Rey final in which Barcelona hammered Vicenzo Montella's side. He has since been sacked with Joaquin Caparros replacing him and the side from beside El Nervion responded to his coaching thus far. They beat Real Sociedad last weekend and look to continue their fight for a European place.
Zinedine Zidane has opted to rest several of his star players with some of them injured and suspended to help with his rotation. Cristiano Ronaldo, Isco and Dani Carvajal are injured while Gareth Bale is suspended. Marcelo, Varane, Modric, Kroos and Navas are all rested.
Sevilla, on the other hand, have Jesus Navas out injured with Carole, Sarabia and Kjaer joining him on the treatment table. Caparros says before the game that, "We said we had to go step by step. Friday's match is history now. Now another match arrives against a great team. We depend on ourselves."


text:
This package of Singapore Flyer tickets takes you to the amazing Ferris wheel built near the Marina Bay in the very heart of Singapore. Adorning the skyline of Singapore, this was once the tallest Ferris Wheel in the world, with a neck-craning height of 165 metres from the ground. Promoted as an observation wheel, the Singapore Flyer has 28 spacious and air-conditioned capsules for people to get in and enjoy the panoramic views of the city around.
A favourite of all the photography enthusiasts, travellers, sightseers and couples, the Singapore Flyer is a must visit attraction in the city. The lengths that you can see up to from the top will truly dazzle you, creating an all-time favourite memory in your mind. Irrespective of the time you come here, the one guarantee that can be given for this place is a stunning view of the entire island and an unforgettable experience. Although the best time to experience this is during or after sundown, you can take in the views from up there any time during the day. The Ferris wheel cum observation wheel slowly ascends to the top, from where you can see as far as 45 km, and on clearer days, even as far as some parts of Malaysia and Indonesia.
One of the most fun and interesting part of the Flyer is that it was made with such amazing precision so it experiences less or even zero wind resistance even in extreme weather conditions. This makes the entire experience smooth and plain sailing on your worries. You do not have to worry about any adverse weather conditions when on the wheel. To add to the comfort of the visitors, the capsules are built to the size of 4 metres x 7 metres, which are as large as a small bus, and can carry as much as 28 people each.
Once you have boarded and settled in, the next 30 minutes are going to be the most memorable as you slowly ascend to the top, spend some time there and then descend back down, fully pumped and excited about the whole experience. And shall you visit it after sundown, the lights of the Singapore skyline are sure to amaze you with theivibrancyce, especially when you look down at the Marina Bay Sands standing tall and in all their glory?
The attractions of Singapore that you can see from the Flyer are Singapore’s east coast, Marina Barrage, Gardens By The Bay, some parts of Malaysia and Indonesia, Marina Bay Sands, MBFC, National Stadium, Raffles Place, Merlion Park, Empress Palace, Padang, Changi Airport and Sentosa Island. The place is perfect if you are wishing to start or end your stay in Singapore, so you can either look forward to seeing all these places or can relive all the memories that you created there.
These Singapore Flyer Tickets entitle you to a ride on the Ferris Wheel, along with a sightseeing trip of the amazing areas nearby. You will also get a pickup from the hotel, post which a sightseeing trip will follow to take you to visit Merlion Park, Fountain of Wealth, Suntec City, Orchard Road, Chinatown, and Little India, ending the trip with an unforgettable ride on world’s second largest observation cum Ferris wheel, the Singapore Flyer


text:
Everyone Gets a Free Smoothie Today Just for Wearing Sandals
Flip flops and food aren't the most natural partners. However, this is one of the rare instances where you won't feel a little weird about the combination.
Tropical Smoothie Cafe has hosted a National Flip Flop Day even year since 2007, and the 2018 installment lands on Friday, June 15. Drop into one of its more than 600 locations in 44 states and you will get a free Sunshine Smoothie anytime between 2pm and 7pm. The Sunshine Smoothie is a combination of mangos, bananas, oranges, and pineapple. That's so intensely tropical it's possible you'll suddenly find yourself in a cabana shirt making reservations at the nearest Margaritaville.
All you have to do to earn that free smoothie is wear sandals. That's it. Show your toes, and you're all set. It's all part of the Cafe's effort to raise $1 million for Camp Sunshine, a cost-free, year-round camp for kids with life-threatening illnesses and their families. The smoothies are 100% free, but customers are encouraged to contribute a little to Camp Sunshine to help the smoothie shop meet its goal.
"These families have had to endure some of life’s most challenging circumstances, and we’ve seen how much Camp Sunshine means to them," Mike Rotondo, CEO of Tropical Smoothie Cafe, said in a statement. "Some days, you have to create your own sunshine, and that’s the beauty of Camp Sunshine, it creates sunshine for these families."
That's an easy path to feeling good about yourself both because you're helping a family in need and because you just landed a free smoothie. The summer is off to a good start.

domain: news


The following procedure lets you create labels for one or more selected sequences automatically, using specified fields such as sequence length, organism, or description.
- Select one or more sequence names from any MegAlign Pro view.
- Right-click on the selection and choose Rename with Fields, or instead choose Edit > Rename with Fields from the main menu. Making no selection prior to using the main menu command is equivalent to selecting all sequences.
The Choose Label dialog opens with the Display drop-down menu set to One or more fields.
The left pane shows available fields, and the right pane shows fields that will be displayed as part of the sequence name. Initially, Default name is the only field listed in the right pane. An example showing the appearance of the name using the specified field(s) appears in the Example box at the bottom of the dialog.
- Select and organize the fields you wish to display as part of the sequence name:
- To add or remove items, move them between panes using the left and right arrows ( ). Use the up and down arrows on the right ( ) to specify the order in which displayed fields should appear.
- By default, a hyphen (-) is included between each of the displayed fields. If desired, type in a different Separator. Or remove all text in the Separator box to include nothing between the fields.
- The Example text box shows what the label will look like with the current selections. This text can be manually edited, if desired.
- When you are satisfied with the choices made in this dialog, click OK to save changes and exit from the dialog. Or click Cancel to leave the dialog without saving any changes.
Need more help with this?


text:
Background Children with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an anti-GD2 monoclonal antibody, significantly improved event-free survival in children with GD2+ NB post autologous stem cell transplantation and enhanced natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity. Thus, approaches to increase NK cell number and activity, improve persistence and trafficking, and enhance tumor targeting may further improve the clinical benefit of dinutuximab. N-803 is a superagonist of an interleukin-15 (IL-15) variant bound to an IL-15 receptor alpha Su-Fc fusion with enhanced biological activity.
Methods The anti-tumor combinatorial effects of N-803, dinutuximab and ex vivo expanded peripheral blood NK cells (exPBNK) were performed in vitro using cytoxicity assays against GD2+ OS, NB and GBM cells. Perforin and interferon (IFN)-γ levels were measured by ELISA assays. Multiple cytokines/chemokines/growth factors released were measured by multiplex assays. Human OS, GBM or NB xenografted NOD/SCID/IL2rγnull (NSG) mice were used to investigate the anti-tumor combinatorial effects in vivo.
Results N-803 increased the viability and proliferation of exPBNK. The increased viability and proliferation are associated with increased phosphorylation of Stat3, Stat5, AKT, p38MAPK and the expression of NK activating receptors. The combination of dinutuximab and N-803 significantly enhanced in vitro cytotoxicity of exPBNK with enhanced perforin and IFN-γ release against OS, GBM and NB. The combination of exPBNK+N-803+dinutuximab significantly reduced the secretion of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β) from OS or GBM tumor cells. Furthermore, OS or GBM significantly inhibited the secretion of regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α) from exPBNK cells (p<0.001) but significantly enhanced monokine induced by gamma interferon (MIG) secretion from exPBNK cells (p<0.001). N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of OS, GBM or NB xenografted NSG mice.
Conclusions Our results provide the rationale for the development of a clinical trial of N-803 in combination with dinutuximab and ex vivo exPBNK cells in patients with recurrent or metastatic GD2+ solid tumors.
- killer cells
- brain neoplasms
- expanded natural killer cells
- targeted immunotherapy
Data availability statement
Data are available on reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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- killer cells
- brain neoplasms
- expanded natural killer cells
- targeted immunotherapy
Osteosarcoma (OS) is the most common primary bone tumor in children, adolescents, and young adults.1 While the 5-year event free survival (EFS) in patients with localized OS remains around 70%–75%, for patients with metastatic disease at diagnosis and those with progressive or relapsed disease, the prognosis is dismal with <20% EFS.2 Among high-grade gliomas, childhood glioblastoma multiforme (GBM) is the most aggressive and patients’ survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy.3 Pediatric neuroblastoma (NB) is the most common extracranial solid tumor in children, with approximately 800 new cases diagnosed in the USA in 2015.4 Forty-five percent of children with NB have high-risk tumors at diagnosis, for which the 5-year EFS remains <50% despite combination therapy with myeloablative chemotherapy, radiotherapy, stem cell transplantation, isotretinoin, and anti-GD2 antibody immunotherapy.5 6 The prognosis for those with high-risk disease at diagnosis and those who relapse is dismal with <20% 5-year EFS.7 8 Therefore, novel therapies including combinatorial immunotherapy are desperately needed for patients with relapsed or metastatic/refractory OS, GBM and NB.
GD2 is highly expressed on neuroectoderm-derived pediatric tumors and sarcomas, including NB, OS, rhabdomyosarcoma, and Ewing sarcoma.9 10 Dinutuximab is a GD2-binding monoclonal antibody used in combination with granulocyte macrophage colony stimulating factor, interleukin-2 (IL-2) and isotretinoin, for the treatment of pediatric patients with high-risk NB5 and is undergoing investigation in patients with relapsed OS11 (NCT02484443).
Natural killer (NK) cells are an attractive candidate as a cellular therapy approach in patients with a variety of malignancies.12 Unlike T cells, NK cells kill tumor cells in a major histocompatibility complex independent manner without the need for prior sensitization.13 NK cells are easily isolated, expanded ex vivo and can be made available as an off-the-shelf allogeneic product for immediate clinical use in adoptive or autologous cell therapies.12 The barriers to NK cells therapy include small numbers of active circulating NK cells, poor persistence, lack of specific tumor targeting, exhaustion, inhibitory receptor induced inhibition, and poor trafficking and tumor infiltration.12 14 Our group and others have successfully expanded active NK cells in vitro by short-term culture with cytokines alone and coculture with engineered feeder cells.15 16 We have demonstrated that expanded peripheral blood NK cell (exPBNK) targeting specificity can be enhanced by engineering exPBNK cells to express chimeric antigen receptors (CAR) such as an anti-CD20 CAR against CD20+ B cell non-Hodgkin’s lymphoma.17 18
N-803 (formerly known as ALT-803) is a new interleukin-15 (IL-15) superagonist and was developed to increase NK persistence and activation in vivo.19 It consists of an IL-15 superagonist mutein (IL-15N72D) and a dimeric IL-15 receptor alpha (IL-15Rα)/Fc fusion protein (figure 1 (online supplemental file 1)). N-803 has 25 times greater in vivo activity and significantly longer serum half-life as compared with IL-15.20 N-803 is currently being investigated in clinical trials to treat patients with myeloma, melanoma and relapsed hematological malignancies, and is well tolerated, and has no dose-limiting toxicity.21 The efficacy of exPBNK in combination with N-803 and dinutuximab against GD2+ OS, GBM, and NB has not yet been investigated. We, therefore, investigated the combination of N-803, dinutuximab and exPBNK cells against GD2+ OS, GBM, and NB both in vitro and in vivo and the mechanism associated with this combinatorial immunotherapy.
Materials and methods
Cell lines and reagents
U2OS (OS), M059K (GBM) and SKNFI (NB) cell lines were purchased from the American Type Culture Collection, Gaithersburg, Maryland. K526-mbIL21-41BBL cells were generously provided by Dean A. Lee, MD/PhD from Nationwide Children’s Hospital, Columbus, Ohio.22 Dinutuximab was generously provided by United Therapeutics, Silver Springs, Maryland. N-803 was generously provided by Hing Wong, PhD, Peter R. Rhode, PhD, John H. Lee, MD, and Jeffrey T. Safrit, PhD from ImmunityBio/Altor Bioscience, Culver City, California. Leukocytes were obtained after informed consent from healthy donors at the New York Blood Center, New York, New York. Peripheral blood mononuclear cells (PBMNCs) were obtained by Ficoll gradient (Amersham Biosciences, Piscataway, New Jersey, USA) separation as we previously described.17 U2OS, M059K and SKNFI cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics penicillin and streptomycin (100 µg/mL). K526-mbIL21-41BBL cells were cultured in complete medium (RPMI1640 medium supplemented with 10% FBS and penicillin and streptomycin (100 µg/mL)). NK cells were cultured in complete medium with 50 IU IL-2.
NK cell expansion
PBMNCs were stimulated with irradiated genetically modified K562-mbIL21 - 41BBL cells for 2 weeks as we previously described.22 Expanded PBNK cells were isolated by negative selection using Miltenyi NK cell isolation kit (Miltenyi Biotec, Cambridge, Massachusetts) as we have previously described.17
Bioluminescence based in vitro cytotoxicity
Bioluminescence (BLI) based in vitro cytotoxicity assays were performed as previously described with minor modification.23 Luciferase-expressing tumor cells were placed in 96–well flat bottom plates at a concentration of 3×105 cells/mL. Subsequently, effector cells were added at different effector-to-target (E:T) ratios with or without N-803 and dinutuximab and incubated at 37°C for 2–3 days. After incubation, the samples in the plates were spun and 75 µg/mL D-firefly luciferin potassium salt (PerkinElmer, Massachusetts, USA) with fresh media added to the cell pellets in each well. BLI was measured with a luminometer (Molecular Devices Multifilter F5 plate reader) as relative light units (RLU). Cells were treated with 1% Triton X-100 as a measure of maximal killing. Target cells incubated without effector cells were used to measure spontaneous death RLU. Percent lysis was calculated from the data with the following equation: % specific lysis = 100×(spontaneous death RLU – test RLU)/(spontaneous death RLU – maximal killing RLU). All tests were run in quadruplicate.
PBMNCs were stimulated with irradiated genetically modified K562-mbIL21 - 41BBL cells for 2–3 weeks. The same number of purified exPBNK cells were cultured in medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803, the same molar dose of immunoglobulin G (IgG), or medium only for 3 days. CellTiter 96 AQueous one solution cell proliferation assay (Promega, Madison, Wisconsin, USA)18 was used to determine the number of proliferating viable cells following the manufacturer’s instructions. Briefly, 104 NK cells/well were seeded in culture medium containing N-803, IgG or medium at 37°C and 5% CO2. At the end of 3 days incubation, CellTiter 96 AQueous one solution was added to each well and cells were incubated for 4 hours at 37°C and 5% CO2. Finally, spectrophotometrical absorbance was measured using a multifilter plate reader (Molecular Device, San Jose, California, USA) at OD490.
Flow cytometry analysis of intracellular proteins and phosphoproteins
Intracellular proteins and phosphoproteins were measured as we have previous described.18 Fixed and permeabilized cells were stained with fluorescent-dye conjugated anti-human antibodies: phospho-p38 MAPK-PE (ebioscience, #12-9078-41), phospho-Akt1-APC (ebioscience, #17-9715-41), phospho-Stat3-FITC (ebioscience, #11-9033-41), or phospho-Stat5-PE (ebioscience, #12-9033-41). Cells were analyzed using MACSQuant Analyzer (Miltenyi Biotec Cambridge, Massachusetts, USA). No stain, or isotype controls were used for gating.
Bio-Plex Pro human cytokines screening
Cell culture supernatants were collected after 3 days culture and were stored at −80 °C. The concentrations of cytokines/chemokines/growth factors were measured by the Bio-Plex Pro Human cytokines screening panel 48 cytokines assay (Bio-Rad Laboratories, Hercules, California, USA) according to the manufacturer’s instructions. In brief, 50 µL aliquot of sample was diluted 1:4 with sample diluent, incubated with antibody-coupled beads, biotinylated secondary antibodies, and followed by streptavidin-phycoerythrin. The beads were read on a Luminex System (Bioplex 200, Bio-Rad) and the data were analyzed using Bioplex Manager Software.
Enzyme-linked immunosorbent assay (ELISA)
Platelet-derived growth factor (PDGF)-AA (Raybiotech, # ELH-PDGFAA-1), PDGF-BB (Raybiotech, # ELH-PDGFBB-1), interferon (IFN)-γ (eBioscience, # KHC4021), and Perforin (Abcam, # ab46068) concentrations were analyzed by ELISA according to the manufacturer’s instructions. Briefly, recombinant standards were run with serial dilutions. Cell culture supernatants were diluted at 1:1 or 1:4 with assay diluent. 100 uL of diluted samples and standard were added to microwells simultaneously and incubated for 2–2.5 hours at room temperature. Biotin conjugated anti-human PDGF-AA, PDGF-BB, IFN-γ, or perforin antibody was used and incubated for 1 hour at room temperature. After washing, streptavidin-HRP solution was added for 30 min at room temperature. ELISA plates were developed with 100 uL TMB substrate reagents. TMB Stop Solution was added to halt the reaction. The absorbance at 450 nm was measured on a Molecular Devices Multifilter F5 plate reader.
Flow cytometry-based phenotyping of NK activating and inhibitory receptors
The exPBNK cells under different conditions were analyzed for phenotypic expression of inhibitory NK receptors (CD94, NKG2A), inhibitory NK killer cell immunoglobulin-like receptors (KIR) (CD158a, CD158b, CD158e), activating NK KIR (KIR2DSA), activating C-lectin NK receptors (NKG2C, NKG2D), and activating natural cytotoxicity receptors (Nkp46, NKp30, NKp44) by flow cytometry as we have previous described.17 Cells were analyzed using MACSQuant Analyzer (Miltenyi Biotec, Cambridge, Massachusetts, USA). No stain, or isotype controls were used for gating.
Six to eight weeks old NOD/SCID/γ-chain-/- (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). The experimental protocol was conducted in accordance with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at New York Medical College is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Luciferase expressing U2OS-Luc (OS), M059K-Luc (GBM) and SKNFI-Luc (NB) cells were generated as we have previously described.17 4×106 of U2OS-Luc, M059K-Luc cells, or SKNFI-Luc cells were subcutaneously injected in NSG mice on day 0. After confirming the tumor engraftment at day 7, 1×107 exPBNK cells+15 µg IgG, 1×107 exPBNK cells+15 µg dinutuximab, 1×107 exPBNK cells+0.2 mg/kg N-803, 1×107 exPBNK cells+15 µg dinutuximab +0.2 mg/kg N-803, 15 µg dinutuximab +0.2 mg/kg N-803, or phosphate-buffered saline (PBS) was intraperitoneally injected to each mouse. NK cells were administered once a week for 3 weeks and IgG, dinutuximab and N-803 were given twice a week for 6 weeks. Tumor engraftment and progression were evaluated using the Xenogen IVIS-200 system (PerkinElmer, Shelton, Connecticut) as we have previously described.17 Tumor size was estimated according to the following formula: tumor size (cm3)=length (cm) × width2 (cm) × 0.5. Mice were followed until death or sacrificed if any tumor size reached 2 cm3 or larger.
Statistical analyses were performed using the InStat statistical program (GraphPad, San Diego, California, USA). Average values were reported as the mean±SEM. Results were compared using the one-tailed unpaired Student’s t-test with p<0.05 considered as significant. Probability of survival in animal studies was determined by the Kaplan-Meier method using the Prism program V.8.0 (GraphPad Software).
N-803 increased the viability and proliferation of exPBNK with enhanced p-Stat3, p-Stat5, pAkt, p-p38MAPK and NK activating receptors
Expanded PBNK cells (exPBNK) were generated by coculturing PBMNCs with irradiated genetically modified K562-mbIL21 - 41BBL feeder cells for 2 weeks and isolated by negative selection using Miltenyi NK cell isolation kit as we previously described.22 To investigate if N-803 stimulates exPBNK cells viability and proliferation as compared with the same molar dose of IgG, the purified exPBNK cells without any feeder cells were cultured in medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803 or molar equivalent dose of IgG for 3 or 7 days. The exPBNK cells with N-803 at 0.35 ng/mL or 3.5 ng/mL had significantly higher viability as compared with IgG or medium controls (p<0.001). Furthermore, N-803 at 3.5 ng/mL significantly stimulated the proliferation of exPBNK cells as compared with N-803 at 0.35 ng/mL at day 3 (figure 1A) (p<0.001) and day 7 (figure 2 (online supplemental file 1)) by MTS assays. We also observed that sustained viability and proliferation of exPBNK cells stimulated by N-803 at 3.5 ng/mL with morphological changes in NK cell shape, size, and number which correlate to NK proliferation and activation as compared with NK cells cultured with IgG (figure 1B). Consistent with enhanced exPBNK proliferation and similar to IL-15, N-803 at 3.5 ng/mL significantly enhanced the phosphorylation of Stat 3, AKT1 and p38MAPK as compared with IgG (p<0.01) at day 3 (figure 1C).
The expression of receptors on viable exPBNK cells was compared by flow cytometry analysis. Purified non-expanded NK (PBNK) cells were used as controls. N-803 at 3.5 ng/mL significantly enhanced the expression of NK activating receptors: NKG2D, NKp30, NKp44, NKp46 at day 10 as compared with IgG (p<0.001) (figure 1D). N-803 at 3.5 ng/mL also significantly enhanced the expression of CD16, known as FcγRIII, at day 10 as compared with IgG (p<0.001) (figure 1D), supporting in part the rationale for the combinatorial immunotherapy of monoclonal antibody with expanded NK cells and N-803.
The combination of dinutuximab and N-803 significantly enhanced in vitro cytotoxicity of exPBNK with enhanced perforin and IFN-γ release against OS, GBM and NB
Since N-803 stimulated exPBNK cells to express high level of CD16 (figure 1D) and dinutuximab is an IgG1 type monoclonal antibody, we investigated whether the combination of N-803 and dinutuximab significantly stimulates the antibody dependent cellular cytotoxicity (ADCC) of exPBNK cells against OS, GBM, and NB. Tumor cell lines: U2OS, M059K, SKNFI cells express GD2 (figure 3 (online supplemental file 1)) and were treated with 1 µg/mL IgG +exPBNK, 3.5 ng/mL N-803 +exPBNK, 1 µg/mL dinutuximab +exPBNK or 3.5 ng/mL N-803 +1 µg/mL dinutuximab +exPBNK cells at E:T ratio=1:1 and 3:1. We found that the combination of N-803, dinutuximab and exPBNK significantly killed U2OS, M059K and SKNFI cells (p<0.001) as compared with other groups (figure 2A) in an E:T ratio dependent manner. The enhanced in vitro cytotoxicity of exPBNK cells was associated with significantly enhanced secretion of perforin (figure 2B) and IFN-γ (figure 2C) from exPBNK cells as compared with all other groups against U2OS, M059K and SKNFI cells (p<0.001) at E:T=3:1.
Cytokines and growth factors screen of exPBNK cells against OS and GBM stimulated by N-803+ dinutuximab
To investigate the cytokines and growth factors that are significantly secreted by exPBNK cells stimulated by N-803 and dinutuximab against tumor cells, exPBNK cells were cultured with N-803, dinutuximab or the combination of N-803 and dinutuximab with or without U2OS cells at E:T=3:1 for 3 days. The concentrations of cytokines/chemokines/growth factors in the supernatants were measured by the Bio-Plex Pro Human cytokines screening panel 48 cytokines assay. For the factors that are secreted by U2OS tumor cells such as tumor factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β), the combination of exPBNK +N-803+dinutuximab significantly reduced the secretion of these factors from U2OS tumor cells as compared with U2OS alone (p<0.001) and exPBNK +U2OS (p<0.01, p<0.05 and p<0.001, respectively) (figure 3A). For the factors that are secreted by exPBNK cells and can be further enhanced by N-803 and dinutuximab such as regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α), U2OS significantly inhibited the secretion of these factors from exPBNK cells (p<0.001) (figure 3B). Monokine induced by gamma interferon (MIG) and interferon gamma-induced protein 10 (IP-10) are important ligands of CXCR3 that are pivotal for NK-cell migration towards tumor cells.24 We found that without U2OS tumor cells, exPBNK cells or exPBNK cells incubated with N-803 +dinutuximab do not secrete MIG. However, U2OS significantly increased the secretion of MIG and IP-10 from exPBNK cells (p<0.001) (figure 3C). Macrophage inflammatory proteins (MIP) 1 alpha and beta are members of the C-C motif subfamily of chemokines and both are ligands of C-C chemokine receptor type 5 receptor, which is essential for NK trafficking in host defense.25 We found that the combination of U2OS, N-803 and dinutuximab significantly enhanced the secretion of MIP-1beta from exPBNK cells (p<0.001) (figure 3D), while MIP-1alpha secretion by exPBNK cells was significantly enhanced by U2OS alone, N-803+ dinutuximab, or U2OS+N-803+dinutuximab (p<0.001) as compared with exPBNK cells (figure 3E).
We further confirmed our findings using M059K cells as tumor targets. We found that the combination of exPBNK +N-803+dinutuximab reduced the secretion of TRAIL (p<0.05), and significantly reduced the secretion of PDGF-BB (p<0.01), and SCGF-β (p<0.05) from M059K tumor cells as compared with exPBNK +M059K (figure 4A). M059K significantly inhibited the secretion of SDF-1α from exPBNK cells (p<0.001) (figure 4B) but significantly enhanced MIG secretion from exPBNK cells (p<0.001) (figure 4C). The combination of M059K, N-803 and dinutuximab significantly enhanced the secretion of MIP-1beta from exPBNK cells (p<0.001) as compared with all other groups (figure 4D).
PDGF-BB is one of the five ligands of PDGF Receptor (PDGFR) α and PDGFRβ. PDGFRs and their ligands have been found to be overexpressed or mis-regulated in many cancers, such as gliomas and sarcomas26 and PDGFRs are also expressed on the non-cancerous cells of the tumor microenvironment to support the growth of cancer cells.27 These findings were similar when we assessed PDGF-AA that the combination of exPBNK +N-803+dinutuximab significantly reduced the secretion of PDGF-AA (p<0.001) from U2OS or M059K tumor cells as compared with the NK, NK +N-803, or NK +dinutuximab (figure 4E).
N-803 combined with dinutuximab and exPBNK cells significantly inhibited OS cells growth and extended the survival of OS xenografted NSG mice
To investigate if N-803 stimulates the proliferation of exPBNK in vivo and has anti-tumor effect with exPBNK cells, we generated luciferase expressing U2OS-Luc cells and xenografted U2OS-Luc cells to immunodeficient NSG mice.18 We found that the mice treated with exPBNK cells+N-803 have a significantly higher number of human NK cells as compared with the mice treated with exPBNK alone (figure 5A). Tumor burden was significantly reduced in mice treated with exPBNK cells+N-803 as compared with mice treated with exPBNK cells alone (figure 5B).
We further confirmed the anti-tumor effects of N-803 combined with dinutuximab and exPBNK cells in human U2OS cells xenografted NSG mice. The U2OS xenografted mice treated with exPBNK cells+dinutuximab + N-803 had significantly smaller tumor sizes (figure 5C) and BLI signals than other groups (figure 5D), and significantly longer survival (figure 5E).
N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of GBM and NB xenografted NSG mice
To confirm that the effect of combination therapy was not specific to OS disease, we investigated if the combination of N-803 with dinutuximab and exPBNK significantly enhances the overall survival of NSG mice with GBM or NB diseases. NSG mice were xenografted with tumor cell line M059K or SKNFI. We found that the combination of exPBNK cells+dinutuximab + N-803 (n=7) significantly extended the survival of M059K mice as compared with the control groups which were treated with PBS (n=4, p<0.001), exPBNK +IgG (n=5, p<0.001), exPBNK +N-803 (n=5, p<0.001), exPBNK +dinutuximab (n=4, p<0.001), and dinutuximab +N-803 (n=4, p<0.01) (figure 6A). Moreover, the combination of exPBNK cells+dinutuximab + N-803 (n=8) significantly extended the survival of SKNFI mice as compared with the control groups which were treated with PBS (n=6, p<0.001), exPBNK +IgG (n=5, p<0.01), exPBNK +N-803 (n=7, p<0.01), exPBNK +dinutuximab (n=7, p<0.05), and dinutuximab +N-803 (n=6, p<0.01) (figure 6B).
Recently, there has been a significant increase in development of targeted cancer therapeutics, particularly against hematologic malignancies. However, the success in terms of developing novel therapeutics against solid tumors is still lagging. In this study, we demonstrated that combining dinutuximab with N-803 significantly enhances the cytotoxic potential of NK cells against NB, OS and GBM in vitro, and significantly improved the survival of NB, OS and GBM xenografted NSG mice.
N-803 has been shown to be a promising therapeutic agent in phase 1 clinical trials, resulting in a significant increase in number and function of NK cells, with an excellent safety profile.21 Consistent with those results, our data shows that N-803 significantly stimulated the proliferation of exPBNK cells (figure 1A,B). NK cells have been shown to exert their cytolytic effects using downstream signaling involving p38MAP kinase and JNK MAP kinase pathways.28 29 Using phosphoflow analysis, we showed that both IL-15 and N-803 significantly enhanced the proliferation of Stat3, AKT1 and p38MAP kinase (figure 1C), consistent with the increased proliferation of exPBNK cells. NK cells express a variety of activating and inhibitory receptors, and the net functionality of NK cells is a complex interplay of signals between activating and inhibitory receptors.30 We have previously shown that expression of activating NK cell receptor ligands (MIC A/B) on malignant cells mediates the improved cytotoxicity of NK cells by engaging with activating NK cells receptors.31 We have also shown in our previous studies that there is high expression of NKG2D on our ex vivo expanded PBNK cells.17 More recent studies have shown that decreased expression of the activating receptors NKp30, NKp46, NKG2D, and DNAM-1 on the peripheral NK cells was positively associated with tumor progression.32 We have shown that N-803 significantly enhanced the expression of NK activating receptors NKG2D, NKp30, NKp44 and NKp46 (figure 1D) and thereby enhancing the cytolytic potential of exPBNK cells. CD16 (FcγRIII) can trigger NK-mediated ADCC.33 The enhanced expression of CD16 on exPBNK cells by N-803 provides the rationale for the combinatorial therapy of exPBNK cells with N-803 and dinutuximab. Additionally, we observed that N-803 maintained the high expression levels of CD94, KIR2DL1 and KIR2DL2/3 on exPBNK cells (figure 1D) during coculture. CD94 forms a heterodimeric inhibitory receptor with NKG2A, and activating receptors with NKG2C, and E in humans.34 A recent study showed that the expression of CD94 on ex vivo-differentiated NK cells was associated with higher lytic potential, and higher ability to form immunological synapses with leukemic target cells.35 The human KIR are key regulators of the development, maturation, tolerance and activation of NK cells through a process termed as ‘education’, ‘licencing’, or ‘arming’.36 However, the specific roles of the enhanced CD94 and inhibitory KIRs by N-803 in NK cell function remains to be discovered.
Previous studies have demonstrated the efficacy of N-803 as an immuno-stimulatory molecule in its ability to further potentiate the immune effector cells either alone, or when combined with other therapeutic agents like monoclonal antibodies or immune checkpoint blockage agents.37 This further provided us with rationale of combining IL-15 superagonist with dinutuximab in an attempt to enhance the cytoxicity against GD2 positive solid tumors. We showed that the combining N-803 with dinutuximab had significantly higher in vitro cytotoxic potential against NB, OS and GBM cell lines as compared with N-803 and dinutuximab alone (figure 2A). NK cells exert their effector function by promoting the granule exocytosis pathway where perforin and granzyme B from the granules are released on conjugation with the target cells.38 However, it has been well published that NK cells undergo exhaustion and subsequently a reduction in granzyme B and perforin levels on serial contact with the target cells.39 Our data suggests that in large part the combination of N-803 and dinutuximab significantly enhanced the secretion both perforin (figure 2B) and IFN-γ (figure 2C) from exPBNK cells compared with all other groups against U2OS, M059K and SKNFI. These data suggest that increased cytotoxicity could in part be due to the ability of N-803 to overcome this NK cell ‘exhaustion and anergy’ and thereby providing a prolonged NK cell ADCC effect resulting in a more robust killing of tumor cells when combined with anti GD2 antibody in the presence of N-803.
It has now been well understood that the tumor microenvironment aids in the growth and survival of tumors in part by inhibiting the immunologic response.40 There has been a significant improvement in understanding of factors that can lead to a diminished efficacy of NK cell based targeted therapies against solid tumors. These factors include but are not limited to poor trafficking and infiltration into the tumor, increase in angiogenesis, downregulation of activating receptors on the effector cells and the presence of chronic immunosuppressive signals in the tumor microenvironment thereby resulting in inhibition of NK cell function and activity.14 41 Even though TRAIL induces apoptosis in some tumor cells and was considered for a potential use in anti-tumor therapy, several lines of evidence demonstrated that TRAIL induced the growth and proliferation of some tumor cells in vitro and promoted the proliferation and metastasis of tumor cells in mice.42 43 The PDGF factors and their receptors (PDGFRs) play important roles in oncogenesis, drug resistance, and are associated with clinical cancer recurrence.44 45
We showed that combining N-803 with dinutuximab significantly decreased the secretion of TRAIL and PDGF-BB (figures 3A and 4A), thereby overcoming some of the immunosuppressive effects of the tumor microenvironment and potentially preventing tumor relapse and metastasis. SCGF-β has been shown to be a predictor of responsiveness to therapy in certain solid tumors.46 We also showed that the combination of N-803 and dinutuximab significantly decreased the secretion of SCGF-β (figures 3A and 4A), partially contributing to the superior in vitro and in vivo anti-tumor efficacy of the combination therapy of exPBNK with N-803 and dinutuximab. RANTES is known as CC chemokine ligand 5. RANTES mediates the trafficking and homing of lymphoid cells such as T cells and NK cells by binding to G-protein-coupled receptors and induces the activation and proliferation of NK cells.47 48 However, RANTES in the tumor microenvironment also plays diverse roles to favor tumor survival and metastasis.49 50 Similarly, SDF-1α, known as CXC chemokine ligand-12, is another factor that is important for NK development and homing51 and but also has controversial functions in regulation of tumor growth, metastasis, and development of chemoresistance after binding to its receptor C-X-C chemokine receptor type 4 (CXCR4).52 We found that OS or GBM tumor cells significantly inhibited the secretion of RANTES and SDF-1α from exPBNK cells (p<0.001) (figures 3B and 4B). Further investigation is needed to unveil if the reduction of RANTES and/or SDF-1α creates pro- or anti-tumor microenvironment. Notably, increased secretion of MIG, IP-10 and MIP-1α and 1β likely resulted in improved trafficking of NK cells into the tumor tissues. In our in vivo animal model, the combination of dinutuximab with N-803 and exPBNK cells resulted in a superior survival benefit and a decreased tumor burden in an OS xenografted mouse model. We confirmed our findings in both a GBM and a NB xenografted mouse models that the combining N-803 with dinutuximab and exPBNK cells had superior in vivo antitumor efficacy.
In conclusion, these results demonstrate that there is significant improvement in the anti-tumor activity, both in vitro and in vivo, when N-803 is combined with dinutuximab as compared with either treatment group alone. Our preclinical data provides compelling evidence that the combination of N-803 and anti-GD2 monoclonal antibody dinutuximab with or without exPBNK would be a reasonable and potentially promising approach for designing future clinical studies against relapsed and refractory GD2 positive solid tumors.
Data availability statement
Data are available on reasonable request.
NSG mice were bred, treated, and maintained in the animal facility of New York Medical College with NYMC International Animal Care and Use Committee approved protocols. The animal experiments were conducted in accordance with the recommendations of the Guide for Care and Use of Laboratory Animals.
The authors would like to thank Erin Morris, BSN and Virginia Davenport, RN for their excellent assistance with the preparation of this manuscript. The authors would like to thank Dean A. Lee for providing the K562-mbIL21-41BBL cells and Hing Wong, Peter R. Rhode, Patrick Soon-Shiong, John H. Lee, and Jeffrey Safrit from ImmunityBio/Altor Bioscience for providing N-803, United Therapeutics for providing dinutuximab and Janet Ayello for her assistance with purchasing research reagents.
Presented at Presented in part at BMT Tandem meeting (2019), Houston, Texas and at Pediatric Blood & Marrow Transplant Consortium meeting (2019), New Orleans, LA.
Contributors YC and MSC conceived and designed the study; YC, GN and SJ developed the methodology, performed the analysis and interpreted the data; YC, GN, JMR and MSC wrote, reviewed and revised the manuscript; and PS-S, JTS and DL provided administrative, technical and material support. All authors approved the final manuscript for submission.
Funding The research for this study was supported by the grants from Pediatric Cancer Research Foundation (PI: MSC), New York Medical College School of Medicine Translational Science Institute Children Health Translational Research Award (PI: YC/JMR), Department of Defense W81XWH-16-PRCRP-TTSA #CA160461 (Partnering PI: MSC), and NIH 1U54 CA232561 01A1 (Sub-PI: MSC).
Competing interests MC serves as a consultant for Jazz, Omeros and Novartis and a Speakers Bureau for Jazz, Servier, Amgen, Sanofi and Sobi. DL reports personal fees and other from Kiadis Pharma, CytoSen Therapeutics, Courier Therapeutics, and Caribou Biosciences outside the submitted work; In addition, DL has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. PS-S is a majority shareholder of ImmunityBio, Inc. related to N-803. JTS is an employee of NantKwest, an affiliated company to ImmunityBio, Inc related to N-803. Other co-authors declare they have no conflict of interest.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

domain: medical


The official website of Davis Cup by BNP Paribas features news, live scores, results, videos and photos from the largest annual team competition in sport. Day 4 Saturday. The Irish Davis Cup team have secured promotion to Group II of the Davis Cup by BNP Paribas following a win over hosts. The pairing could not have been better, despite the fact it is being played by two first-round Davis Cup losers. Ireland was beaten by Italy in.
Ireland Davis Cup, Estonia 2016
Davis cup ireland - William Hill
Doyle has been the team's catalyst and, paired with Sorenson in the doubles, he gives the Irish team a respectable chance in almost every series it plays, although he is realistic about the upcoming Davis Cup match with the United States. R in the other play-off. Olympic Games Paralympic Games European Games. Bulgaria sent in their two singles players for the deciding rubber while Ireland brought in doubles specialist David O'Hare alongside Barry. The day started with the resumption of the final pool match against Montenegro which was held over due to failing light on Friday evening. Doyle is one of those 44 million and, to the Irish, it is only fitting that he has reversed the immigration trend somewhat.


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domain: agronomy