Formal funeral for criminal brother?
Our youngest brother has been diagnosed with laryngeal cancer recently. His oncologist has given him 2-4 months and hospice has come in.
Quite frankly, our brother looked forward to hospice, because of 'all the drugs' he would get. He is 55 years old, has never had a job and has made a life of drinking, taking and selling drugs (if he has any left). He's been in prison three times and has spend numerous months in jail. He lives on $750 per month, SSI. He lived off of my parents, when they were alive and has stolen from all his family since he was very small.
The three of us have productive, normal lives. Two of us are in the medical field.
The problem, he now expects US to pay for his formal funeral, as does his live-in girlfriend, with his internment to follow in our family plot. He will not hear of the state's cremating him nor burying him. He feels entitled. The girlfriend is on assistance, too, and is broke.
What the heck do we DO and what do we tell him?
It doesn't matter what you tell him. When the time comes, he won't be around to check up. Just get through the next few months as peacefully as possible. You owe him nothing. Treat any porkies as a different version of the 'therapeutic lie' we all talk about.
The guys dieing at 55 and his self righteous sister wants to keep him out of the family plot. If it really doesn't matter what happens because he's dead anyway why have a family plot to begin with?
Well, I have the same... in the future anyway. One of my brothers is in prison and will not be out until 2027. My other brother is on assistance. I have insurance policies for both enough just to pay for what I think will be necessary.
Tell him you will "take care of it". Promise nothing. No point in arguing. Make no written contract.
Girl friend -- pay for what you can but if you want a major funeral/burial -- better get a job.
Do what you can. If its only what the state can do well that's it.
This is a sad situation but I think you could tell him you'll have a send-off of some kind for him but that under the circumstances it's not going to be an expensive shin-dig. Tell him he's lost the right to be saying things like he won't hear of the state's cremating him or burying him. He lost the right to call the shots when he chose the life style he did.
Entitlement is a silly overused word I M O. Too many people, I don't care who you are, how you look, what you do, no one is really entitled to anything. But you reap what you sow. He should just concentrate maybe on making things right with you guys and his maker instead of worrying about what kind of casket he is going to lay in.
"What the heck do we DO and what do we tell him?"
Tell him: 'We owe you nothing. If anything we do for you, it's out of our compassion for you as a human being."
If you plan to just give him the very basic, then tell him: "We will GIVE you a basic cremation. If you want anything more than that, you will need to pay for it."
And if you want to pay for some of the cost above the basic, tell him: "If you want more than the basic, you can pay x% and we GIVE you the rest."
Tell him: "For once in your life, before you die, stop being a leech, and be responsible. Give us something good to remember you by."
It seems clear that none of you wants to pay for the elaborate funeral he is planning. He can plan all he wants. The least expensive thing would be immediate cremation in alternative container. Go to a funeral home now to check it out. As next of kin, I believe you siblings would have legal say in the arrangements and girlfriend, not being a spouse, has no say at all. If girlfriend wants something different, and is paying for it, I'm sure you would agree to it. If she is not paying, why would she get a say? As to what you tell him, when he brings it up, nod and smile and change the subject. When the time comes, do what you feel is right.
Welp. This may sound harsh. But to heck with the girlfriend...get a job and get then have a say. So sorry, because some people have legitimate issues and need assistance. But if she's not one of those, then let that go. And, harsh again, but maybe tell him nothing. Just deal with this the best you can, and find peace with it. We all have choices to make and there are consequences to the decisions we make. If he is addicted to drugs, not that it's a choice actually, but getting help and changing the way they live is a choice, and maybe he chose not to change. I wish you well. You're in a tough situation.
They don't owe the girlfriend anything. She does not have a relationship with the family.
This thread hits home with me big time! I lost my brother in 2013. He was in his 60's. He had a complicated life. I loved him with all my heart but his behavior caused the entire family so much grief.
When he was 13 he became addicted to heroin. He was the oldest brother, 7 years older than me. It was terribly confusing for me. Back then methadone was used to treat addicts.
My mom would pile us in the car, drive to the clinic and he would walk inside to get his fix while we waited in the car. I will never forget the sad look on my mom's face.
No support groups back then. Lots of arguments at our house. Everything was hush hush.
Several kids told me when I was young that they couldn't play with me because their parents told them I had a 'bad brother'. I would cry because I said to them that I wasn't bad but it didn't matter. They had to obey their parents. I understood that later in life but not as a kid.
Once he had hallucinations where he thought he had growths of some sort on his arms and my parents had to wrestle a knife away from him to keep him from cutting his arms up trying to remove the imaginary growths. That terrified me!
He overdosed on our front porch. I thought he was dead. My younger brother and I ran to tell my mom. A doctor came to our house and helped him. They made house calls back then for emergencies. My brother was able to be revived. Was very scary for us.
I never understood why we had to drive for hours and pick him up from a large building (juvenile detention home) for a day pass and then bring him back. The ones that were closer he ran away from and found friends to hide out with. When I asked mom why he couldn't come home she only told me that he did something bad. I always wondered if I was bad would I end up there too.
Later he committed armed robbery and went to prison for many years. I couldn't bring myself to visit him and see him locked up.
He got HepC. Mom and dad were ailing by then. They asked me to take care of him, doctor appointments, hospital visits, shopping and so forth.
My dad died. Hurricane Katrina hit our city. Mom moved in with us.
He had a horrible motorcycle accident. I was the only one at the hospital for the surgery. He nearly died. He asked me to buy heroin for him. I told him I would never purchase drugs for him. I fell apart and a nurse told me to take care of myself.
I set him up with hospice. He lied about everything to them. He ate the pain pills like candy. I helped him clean his apartment and almost stuck myself on one of his dirty needles. I finally told my mom I couldn't help him anymore.
I had tried to get him to go to rehab. I truly tied all I could to help him. His problem was bigger than he was.
He stopped hospice, eventually became homeless. I prayed daily for him.
An old man took pity on him and let him stay in a trailer on his property. When he became very sick he brought him to an end of life hospice program.
I brought my mom to see him. He was nothing but skin and bones. He never apologized for hurting me but three nurses told me that he was sorry and that he loved me. I forgave him completely. I was the last one with him seconds before he died. The nurse called me as we were backing out of the parking lot to say he died.
My mom asked me to make the funeral arrangements. He was cremated and is in our family plot. We had a priest go to hospice and say a graveside service.
I have mixed emotions. I loved him as a brother but I hated having an addict in our family. I have enormous compassion for addicts but there is no denying that it is hell for everyone.
He wasn't a bad kid before that. His best friend asked him to try it because he was lonely. His father was a doctor and was never, ever home, at ball games, nothing and wanted to escape the loneliness and asked my brother to join him. His best friend ended up dead too, couldn't pay his dealers so they beat him to death.
I can relate, do what you feel is best.
And you are one of the reasons I still stay with this site. You have compassion and common sense. That's a rare combination. I'm sure your brother appreciated and loved you. What I've learned about drug addicts is that most are not bad people they do bad things to support their habit. Most do want to quit. Most try several times to get clean. They go to church for guidance, try rehab usually multiple times. Each time they relapse they know their family is going to be upset or worse just wash their hands of them.
The disease of addiction is insidious affecting everyone around the addict. So they start hanging around other addicts who don't judge them.
The OP would benefit greatly by attending Al-Anon (for friends and family of alcoholics) or nar-anon (for friends and family of drug addicts.
She sounded so bitter in her post. She doesn't have to have to have an expensive wedding to have a formal one. But to deny him a place in the family plot borders on hateful. And her little quip about him using hospice to get drugs shows she still thinks he needs to be punished. I can't get over the coldness of her post. The guy has less than six months to live. If she doesn't find a way to forgive him before he dies she will regret it.
In a similar situation I just said No. They were not and still are not happy about it but for me it was the right thing to do.
If this fellow has caused people so much pain, why are they hanging around listening to his plans to abuse from the grave? The family has no obligation to put him in their plot. Who is his executor? They get to make the decisions and pay for it. If that's his girlfriend, great, she can pay from her purse. Otherwise I'd just make sure the company that maintains the family plot knows he is not to be buried there. There's no need to argue with him- you can just say no to the girlfriend once he's gone. We've chosen body donation which is better than free as the company will scatter cremains over the Pacific. She's free to do that as well, which is not done by the state.


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Saved By Stupidity
Category: Fine Art Prints
2019, Arcadia Art Gallery
2 color Screen print, signed and numbered by Anthony Ausgang
Edition of 99 with 5 APs
90 x 70 cm. (35.43″ x 27.55″)
Available only from Arcadia Art Gallery
Arcade Art Gallery is pleased to present an exclusive Print Release by established and renowned American Lowbrow and Pop Surrealism artist, Anthony Ausgang.
This print edition was entirely produced in Taiwan, and is a unique collaboration between Arcade Art Gallery and local print masters, Tsubaki Print Studio.
Anthony Ausgang is an original pioneer of the American Lowbrow and Pop Surrealism art movements. Although based in California, he is an established International artist with many successful gallery and museum shows in Japan, Europe and Australia. He also creates concert posters and album artwork for bands and musicians such as, Sonic Boom, MGMT, Green Day, Luna, Neil Young, and the Boredoms.
Arcade Art Gallery and Anthony Ausgang have teamed together to produce, for the first time outside of the United States, an exclusive Collectors' Item print that brings together his famous Low Brow cartoon cat characters and California Hot Rod Culture
With the support of our talented Taiwanese partners, Tsubaki Print Studio, we have produced a beautiful two-colour, hand-pulled, silk screen print on cotton rag paper.
The print is a large format, masterfully executed work of art, and its incandescent hand-pulled color echoes Ausgang's canvas masterpieces, which often have a trademark luminescent finish.
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Home iPhone 15 Best News Apps for Your iPhone and iPad
15 Best News Apps for Your iPhone and iPad
Ravish Sundriyal
Live in the present with the best news apps for your iPhone and iPad.
Digital revolution has transformed the entire broadcast and information industry. There are a lot of things happening in the world, and now you don't have to wait for tomorrow's news to get the update. Everything is working in real time now.
Every media/new company has gone digital created customised apps for the users to feed them with news every minute. You can get live updates from all across the world with just a tap of the screen on your phone.
Today, we will be featuring 15 best news applications for your iPhone and iPad. So, sit tight and pick your favourite one for your daily news updates.
1 – Flipboard
2 – BBC- News App
3 – Google News
4 – New York Times
5 – CIRCA
6 – Pulse
7- Pocket
8 – News 360
9 – BuzzFeed News
10 – Smart News
11- Reddit
12 – Inshorts
13 – News Break
14 – Apple News
15 – Feedly
Flipboard
Developer: Flipboard Inc.
One of the most beautiful looking apps out there. Flipboard is a free new app for your iPhone and iPad.
The layout is pleasing to eyes, the video and article placement is amazing, and you get data from social media channels like Facebook, Instagram and Twitter. Overall, if you are looking to consume news in style, Flipboard is the one.
BBC News
Developer: BBC Worldwide
When it comes to credibility, the BBC is one of the oldest new sources for the world. BBC has its News app for iOs called BBC-News App.
It has all news aligned properly category wise, and there is a section to stream live news as well. The app is fluid and dynamic with its signature colour of red and white. BBC News has given the world the most accurate reporting since its inception.
Google News
Google upgraded its earlier product – Google News Stand to Google news, and it has top-notch features.
You can set up daily updates with Google recommendation to experience a smooth use of the app. It also allows you to save/download/read it later option to make your experience hassle-free. Google News is one of the best news apps for the iPhone and iPad out there.
The New York Times
Developer: The New York Times Company
NY Times is another credible new app out there. Like any other news app, you get all the decent features to read and watch the news.
One unique thing about the New York Times app is the augmented reality feature that lets you experience the content on another level. However, it is a paid element.
Circa – Experience News
Developer: Circa 1605, Inc.
Circa is relatively a new app in the market, but they are already making head turns with the users. Apart from its amazing app interface, they don't just pull news from the web and serve you.
Their in-house team of editors picks and recreate news in a short format which is easy to consume. They also customised the headlines, quotes to make it more impactful.
If you are bored of old-school news format, CIRCA is the best app out there to experiment. One cool thing about CIRCA is that it is one of the few apps that are free out there.
Pulse by Zerodha
Developer: Zerodha Broking Limited
Pulse is similar to Flipboard when it comes to the app structure. However, it is one of the apps that allows you to read news pieces by selecting the source.
Yes, that is an amazing feature to have as you might prefer various source to grab the news. There are a lot of category leaders that provide the best news and articles.
This app lets you select that. Pulse is a free app for the iPhone and iPad.
Pocket
Developer: Read It Later, Inc
If you are a person who wants to save articles and news for later, then the Pocket is the perfect app for you.
The pocket is a minimalistic news app for your iPhone and iPad that allows you to do that. With its clean interface, your reading experience is enhanced.
The app was not found in the store. 🙁
Go to store Google websearch
If you are someone who loves platform integration, then News 360 is the best news app out there for your iPhone and iPad.
It allows you to connect all your social media accounts along with iMessage and various apps.
Apart from integration, it also allows you to select your favourite categories to consume content.
BuzzFeed is one of the world's top content creators. You won't find anyone that doesn't know about BuzzFeed.
You might be surprised to know apart from their entertainment section they also offer global news.
Yes, you can download BuzzfeedNews for your iPhone and iPad and enjoy similar interface like BuzzFeed. Buzzfeed is known for the semi-formal approach which makes them easy and fun to use.
Smart News is a top news app for the iPhone and iPad. It has a larger user base. Apart from having all the relevant news app features app, it has a unique feature that helps users.
Its ability to categorised the news under very niche sections and providing 1 minute read to its users. Smart news is an amazing news app for iPhone and iPad.
Reddit
Developer: reddit
As we all know how cool is Reddit and everyone is a fan. Reddit app gives you access to the global news at your fingertips.
Its clean section and app structure make an awesome reading experience.
You can upvote the trending stories and news like the original platform that allows you to access the latest and most trending stories online.
Inshorts
Developer: ADA NEWS IN SHORTS PRIVATE LIMITED
Too busy to read long format articles and news. Inshorts gives you a 60-word summarisation of the news. Well, this is one of a kind news app for iPhone and iPad. Sometimes, you need a quick update on the topic to keep up with the trend.
Well, Inshorts makes sure you consume a lot of information is less time. This is available in Hindi and English and can be downloaded for iOs as well.
News Break is an AI-driven news app. It is an interest-based app that understands what you like. Which categories, format and even the type of stories.
From global news to funny GIFs, this app allows you to consume content which is technically customised for you.
What is better than an app that is made for your device. Apple News is just made for your iPhone and iPad.
You can experience a magazine style news and articles from the world. You can get the app from the Apple store and enjoy Apple's seamless design and interface.
Feedly - Smart News Reader
Developer: Feedly Inc.
The last news app which we are featuring is Feedly. Feedly is so much more than just a news app. It is a planning and organising app as well.
You can beautifully organise all your content online through Feedly. Its sophisticated app lets you create categories as per your liking.
We all are busy and want to keep updated with the news and happenings around us. These apps have made our life simple.
Everything is on our fingertips, and the ease of accessing it has taken the experience to another level. I hope you found these fantastic news apps helpful.
Do share any kick-ass app and we might have missed here in the comments below.
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Think about the last few 20 you've taken, especially this holiday season. How many of these photos were on a traditional point-and-shoot camera (or SLR) versus how many were taken on your smartphone? If you're like most people these days, less were taken on regular cameras.
Last week, the NPD Group, a research firm, released a study that states of all photos taken in 2011, 27 percent were taken on smartphones, up from 17% a year ago. Traditional cameras took 17 percent of the photos, down from 53% the year before. The fact is – as cameras on smartphones continue to advance in image and lens quality, packing around that Canon camera is becoming less common these days.
For me, the smartphone camera revolution happened back in 2007 when I first utilized the Nokia N95. This phone, advanced for its time and still impressive today, had a 5 megapixel camera with Carl Zeiss lens, resulting in great images and fluid 30 frames-per-second video. Today, I carry a Nokia N8 which has a 12 megapixel shooter and xenon flash (the same you find on your traditional point and shoots) which takes HD quality video and also can send and receive phone calls too.
Unit sales of cameras are down overall, but it's not all bad news for camera manufacturers. SLR-type cameras with detachable lenses are seeing an upswing in sales. Also, I'm seeing some folks shun using their smartphone as a camera because they want the battery life to be saved for actually making calls and sending messages.
How about you – are you a smartphone picture taker? Sound off below in the comment!


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Rosary Square
<Lourdes
The open space in front of the Rosary Basilica is known as Rosary Square. The entrances to the Crypt and the Upper Basilica, both of which are built on top of Massabielle, are far above ground level. To facilitate access, two enormous ramps were constructed, which curve down either side of Rosary Square. The image of the entrance of the Rosary Basilica, flanked by the two ramps and surmounted by the spires of the Upper Basilica, has become one of the iconic symbols of Lourdes, and a stylised form of this image has been adopted by the Domain itself as its logo.
The Statue of the Crowned Virgin, often known as the "Crowned Statue" – La Vierge Couronnée, stands across Rosary Square from the Rosary Basilica and faces the entrance. This prominent statue is a familiar landmark and a traditional meeting point. The statue is 2.5m high and cast in bronze, painted white and blue in the traditional colours. Her rosary is of the Birgittine style and incorporates six decades. Behind the Crowned Statue is the Esplanade, a large open walkway which is used in the processions.


Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-γ and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.
The immune system can recognize and reject primary developing tumors, a concept known as cancer immunosurveillance. In melanoma, cancer immunity exists because the immune system's repertoire contains autoreactive T and B cells that, when activated properly, may reject malignant cells that express unaltered or altered self-Ags (1). Because tumor Ags are often self-Ags, high-affinity T cells are either deleted in the thymus, remain ignorant, or become tolerant in the periphery, probably due to a population of CD4+CD25+ immunosuppressor regulatory T cells (Treg)3 (2). In mouse models, Ab depletion of Treg using anti-CD25 and anti-CD4 Abs has been demonstrated to increase rejection against chemically induced tumor and enhance vaccination against melanoma differentiation Ags (3, 4, 5, 6, 7, 8). Still, depletion of CD4+ and/or CD25+ T cells has little effect on the rejection of spontaneous tumors and may have a detrimental effect on the generation of CD8+ antitumor immunity (6). Other groups have shown a new strategy to inhibit Treg suppression, via the stimulation of glucocorticoid-induced TNF receptor (TNFR) family related protein (GITR), a member of the TNFR superfamily that is constitutively expressed at high levels on Treg (9, 10, 11, 12). GITR expression is not restricted to Treg, and recent studies have shown that although GITR stimulation inhibits Treg suppression (9, 10, 13), it also stimulates CD4+ and CD8+ T cells (12, 14, 15, 16, 17, 18). GITR stimulation has been shown to induce tumor rejection in a concomitant immunity model (19), and expression of GITR ligand on tumor cells can delay tumor growth and increase T cell infiltration (20). Therefore, we tested whether in vivo stimulation of GITR can break tolerance to differentiation Ags and enhance antitumor immunity to B16 melanoma.
C57BL/6, BALB/c, Rag 1−/−, and IFN-γ−/−, pfp−/−, and gld mice (females, 8–10 wk old) were obtained from The Jackson Laboratory. All experiments were performed in accordance with our institutional guidelines. The B16F10 (B16) mouse melanoma cell line was originally obtained from I. Fidler (MD Anderson Cancer Center, Houston, TX) (21, 22) and passaged intradermally to ensure reproducible and aggressive tumor growth. TGL tumor lines (B16-TGL and RENCA-TGL) were generated by transducing B16 and RENCA with a retroviral vector containing a fusion reporter gene coding for HSV1-TK, enhanced green fluorescent protein, and firefly luciferase (23). After transduction, individual clones with high enhanced green fluorescent protein expression were sorted into 96-well plates using a FACSVantage DiVa (Becton Dickinson) cell sorter. Tissue culture medium consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine.
C57BL/6 mice were challenged by intradermal inoculation of 3 × 104 or 1.2 × 105 (high dose) B16 cells, and tumor diameter was measured every 2 days. Mice rechallenged received an intermediate dose of B16 cells (6 × 104 cells). For tumor-free Kaplan-Meier curves, a mass of 2 × 2 mm was considered as (+) for tumor. For bioluminescence experiments, C57BL/6 or BALB/c mice received 1 × 105 cells of B16-TGL or RENCA-TGL i.v. and were given 150 mg/kg i.p. of D-luciferin (Xenogen). Fifteen minutes after D-luciferin injection, mice were anesthetized with isoflurane and placed into the Xenogen IVIS bioluminescence imaging system (Xenogen) in a supine position and recorded for 5 min. Pseudocolor images showing the whole body distribution of bioluminescent signal were superimposed on the conventional grayscale photographs.
C57BL/6 mice were intradermally challenged with 3 × 104 B16 cells and treated with 1 mg/mouse of rat IgG control Ab (n = 50 mice) or DTA-1 (n = 50 mice) after 1, 4, and 9 days from challenge. Twenty-one days after challenge, spleens and draining inguinal lymph nodes were harvested and pooled. CD8+ cells and CD4+ were positively selected using MACS beads (Miltenyi Biotec). Purified cells (>98% purity) were adoptively transferred into Rag 1−/− hosts. Groups of 8–10 mice received either CD4+ cells alone (24 × 106 cells/mouse), CD8+ alone (12 × 106), or CD4+ and CD8+ at a 1:2 ratio (12 × 106 CD8+ and 24 × 106 CD4+ cells per mouse). One day after adoptive transfer, Rag 1−/− mice were challenged intradermally with 3 × 104 B16 cells, and tumor diameter was measured every 2 days.
Mice received 1 mg/mouse of affinity-purified DTA-1 mAb (9) or rat IgG control Ab (Anogen) injected i.p. at the specified time points. Mice were depleted of Gr-1+ (clone Rb6-8C5), CD4+ (GK1.5), CD8+ (2.43), and NK1.1+ (PK136), and CD25+ cells (PC61) by i.p. injection of 500 μg of the mAbs (bioreactor supernatants) at days −7, −4, +4, and +7 from tumor challenge. For depletion of Treg before tumor challenge, mice were depleted with 500 μg of PC61 by i.p. injection at days −7 and −4. Flow cytometry was used to confirm >98% depletion of target cells for at least 7 days after the first injection.
Anti-mouse CD16/CD32 FcR block (clone 2.4G2) and all of the following fluorochrome-labeled and purified Abs against murine Ag were obtained from BD Biosciences: CD4 (clone RM4-5), CD8 (53-6.7), CD62L (MEL-14), CD122 (TM-B1), CD44 (IM7), CD45R/B220 (RA3-6B2), Gr-1 (RB6-8C5), CD25 (PC61), CD69 (H1.2F3), isotype controls: rat IgG2a- (R35-95), rat IgG2a- (B39-4), rat IgG2b (A95-1), hamster IgG group 1 liter (Ha4/8), streptavidin-FITC, -PE, and -PCP. Biotinylated anti-murine GITR (BAF524) was obtained from R&D Systems. T cells were washed in PBS with 2% FBS and 0.1% sodium azide and incubated for 15 min at 4°C with anti-CD16/CD32 FcR block. Subsequently, cells were incubated for 30 min at 4°C with Abs and washed twice. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) with CellQuest or Flowjo software (Tree Star). For detection of FoxP3+ cells (eBioscience), splenocytes and draining lymph nodes were harvested, counted, and 10 × 106 cells were incubated in 2% paraformaldehyde at 37°C for 30 min, washed three times with PBS, and fixed in 80% methanol at −20°C overnight. Cells were washed three times with PBS and incubated with anti-CD16/CD32 FcR block for 15 min. Subsequently, cells were incubated with Ab against cell surface markers, incubated for 30 min at 4°C, washed twice in PBS, and analyzed by flow cytometry.
Peptides analyzed were synthesized by Genemed Synthesis and used at >80% purity, as confirmed by HPLC. Peptides tested include the following: gp100/pmel 17 peptide gp10025–33 (24), dopachrome tautomerase/tyrosinase-related protein 2 (DCT) DCT181–188 (25) and DCT363–370, and TYRP1/gp75 (gp75), gp75455–462, gp75481–489, and gp75522–529 (J. A. Guevera-Patino, M. E. Engelhorn, M. J. Turk, C. Liu, F. Duan, A. D. Cohen, T. Merghoub, J. D. Wolchok, A. N. Houghton, submitted for publication.) For ELISPOT analysis (19), multiscreen-IP plates (Millipore) were coated with anti-mouse IFN-γ Ab in PBS (clone AN18; Mabtech), incubated overnight at 4°C, washed with PBS, and blocked with RPMI 1640 plus 7.5% FBS for 2 h at 37°C. CD8+ T cells were harvested from spleen and inguinal lymph nodes, purified using anti-CD4 and anti-CD8 MACS magnetic beads (Miltenyi Biotec), and plated at a concentration of 2.5 × 105 cells/well. B16 targets cells were pretreated for 24 h with Con A supernatant (T-Stim Culture Supplement Rat with CON A; BD Biosciences), detached using a nonenzymatic method (Cell Dissociation Solution (1×) Nonenzymatic; Sigma-Aldrich), irradiated, and plated at 1 × 104 cells/well. Target EL-4 leukemia cells were pulsed with 10 μg/ml peptide for 1 h and plated at 2 × 104 cells/well. After 20-h incubation for CD8+ T cells and 48 h for CD4+ T cells at 37°C, plates were washed with PBS plus 0.05% Tween and incubated for 2 h at 37°C with biotinylated Ab against mouse IFN-γ (clone R4-6A2; Mabtech). Spots were counted with an automated ELISPOT reader system with KS 4.3 software (Carl Zeiss MicroImaging).
Log-rank analysis was performed using Kaplan-Meier curves. For all other analysis, nonparametric unpaired Mann-Whitney U test was used.
We analyzed two solid tumor cell lines, the spontaneous melanoma B16 and renal cell carcinoma RENCA, and established that both tumor lines were negative for GITR expression (Fig. 1⇓A). To assess whether in vivo GITR stimulation could result in tumor delay or rejection, the agonist Ab against GITR (clone DTA-1) was injected into BALB/c mice challenged with RENCA-TGL (Fig. 1⇓B). Mice that received a single dose of DTA-1 the day before the challenge had increased survival when compared with the control group. This antitumor effect was confirmed in C57BL/6 mice challenged with B16 (without TGL) intradermally (Fig. 1⇓, C and D) or by tail vein (B16-TGL) and treated with a single dose of DTA-1 (data not shown).
A single dose of anti-GITR agonist (DTA-1) induces a delay in tumor progression. A, GITR surface expression of B16 and RENCA tumor cell lines (gray histogram, unstained cells; black line, isotype control; red line, DTA-1). B, Kaplan-Meier survival curve of BALB/c mice challenged i.v. with 1 × 105 RENCA-TGL cells and treated with a single dose of rat IgG control Ab or DTA-1 (1 mg/mouse) 1 day before the challenge. C, C57BL/6 mice challenged intradermally with 1 × 105 B16 and treated with a single dose of rat IgG control Ab or DTA-1 (1 mg/mouse) at different time points of tumor challenge. D, The data represent the mean size of intradermal tumors per group at different time points after challenge. All data are representative of two independent experiments (n = 10–15 mice per group).
We analyzed GITR expression on different effector populations of the adaptive and innate immune system (data not shown). GITR was present at low levels on B cells, at intermediate levels on CD4+, CD8+, NK, NKT cells, granulocytes, and macrophages, and at high levels on Treg.
DTA-1 treatment at day +1 reduced tumor growth more effectively than the other regiments, suggesting that costimulation of effector T cells by DTA-1 was more important for tumor rejection than the modulation of Treg. Based on this rationale, we decided to administer DTA-1 three times, starting 1 day after tumor inoculation (Fig. 2⇓). Most mice that were challenged with a high dose (Fig. 2⇓A) or low dose of B16 (Fig. 2⇓B) and treated three times with DTA-1 rejected the tumor. Next, we examined the contribution of Gr-1+, CD4+, CD8+, and NK1.1+ cells in tumor rejection by depleting the host with specific Ab. Gr-1 depletion had no impact on DTA-1-mediated tumor rejection (Fig. 2⇓C), whereas mice depleted of CD4+ cells (Fig. 2⇓D), CD8+ cells (Fig. 2⇓E), or both (Fig. 2⇓F) were unable to reject B16, even in the presence of DTA-1, indicating that T cells are necessary for DTA-1-induced tumor rejection. Mice depleted of NK1.1+ cells (Fig. 2⇓G) developed tumors rapidly, and addition of DTA-1 was associated with only low levels of rejection comparable to the nondepleted rat IgG control group, indicating that NK/NKT were also required for B16 rejection, which is consistent with other published experiments (26, 27). Prior studies have indicated that anti-CD25 Ab depletion of Treg can enhance antitumor immunity against highly immunogenic tumors but has little or no effect against nonimmunogenic tumors (3, 4, 5, 6, 7). Experiments treating B16 with anti-CD25 administration range from a delay in B16 progression without rejection (7) to low rate rejection (3). Our results show a delay in B16 progression in CD25-depleted mice compared with the control group, although this result did not reach statistical significance. Addition of DTA-1 to anti-CD25 did not result in improved tumor protection (Fig. 2⇓H). Because the presence of the Ab can be detected up to 15–21 days after the first injection (3), these results are consistent with abrogation of immunity due to a block in IL-2 signaling by the anti-CD25 Ab (28, 29, 30) or, alternatively, depletion of recently activated effector cells (6). Rag 1−/− mice that lack T and B cells, but have increased NK activity, were challenged with B16 and treated with DTA-1 or control Ab (Fig. 2⇓I). No difference in tumor take was observed between these two groups. Overall, these results confirm that both T and NK cells are required for tumor rejection.
Three doses of DTA-1 induce B16 rejection that requires T cells and NK/NKT cells. Kaplan-Meier tumor-free curves of C57BL/6 mice treated with control Ab or DTA-1 after intradermal B16 challenge. All mice received 1 mg/mouse of DTA-1 Ab (DTA-1 treatment) or rat IgG control Ab (Rat IgG treatment) on days +1, +4, and +9 after challenge. A, Mice were challenged with B16 at a high dose (1.2 × 105 B16 cells). B, Mice were challenged with B16 at a low dose (3 × 104 B16 cells). C–H, Control groups included mice challenged with B16 (3 × 104 cells) and treated with DTA-1 or control Ab (DTA-1 treatment and Rat IgG treatment). Depletion groups included mice challenged with B16, treated with DTA-1 or rat IgG Ab, and also depleted of the following: Gr-1+ (C), CD4+ (D), CD8+ (E), CD4+ and CD8+ (F), NK1.1+ (G), and CD25+ (H). Depletions were done by i.p. injection of 500 μg Ab/mouse at days −7, −4, +4, and +7 of tumor challenge. These groups are designated as Depletion + rat IgG and Depletion + DTA-1. I, Rag 1−/− mice were challenged with B16 (3 × 104 cells) and treated with DTA-1 (DTA-1 treatment) or rat IgG control Ab (Rat IgG treatment). All data are representative of at least two independent experiments (n = 10–20 mice per group).
To discriminate between the effects of DTA-1 on Treg from that on effector T cells CD25 depletion was done twice, at 7 and 4 days before tumor inoculation (Fig. 3⇓A). Mice depleted of CD25+ cells and treated with control rat IgG developed tumors at the same rate as nondepleted mice treated with the control Ab. All mice depleted of CD25+ and treated with DTA-1 rejected tumors, whereas 80% of nondepleted mice treated with DTA-1 rejected tumors. Although CD25 depletion and DTA-1 treatment seemed to be enhancing tumor rejection, the comparison between both groups did not reach statistical significance. These results indicate that DTA-1 rejection of B16 is dependent on effector T cells rather than abrogation of Treg suppression.
DTA-1-induced rejection of B16 is predominantly mediated by costimulation of effector cells rather than modulation of Treg. Kaplan-Meier tumor-free curves of C57BL/6 mice treated with control Ab or DTA-1 after intradermal B16 challenge. All mice received 1 mg/mouse of DTA-1 Ab or rat IgG control Ab on days +1, +4, and +9 after challenge. A, Control nondepleted mice were treated with DTA-1 or rat IgG Ab (Rat IgG treatment or DTA-1 treatment). Two groups of mice were depleted of CD25+ cells by injection of 500 μg of Ab per mouse 7 and 4 days before B16 challenge. One group received DTA-1 (CD25 depletion + DTA-1), and the other group received the control Ab (CD25 depletion + rat IgG). All mice were challenged intradermally with 3 × 104 B16 cells. B, Adoptive transfer of T cells from tumor-bearing DTA-1-treated mice protect Rag 1−/− mice from B16 challenge. C57BL/6 mice were challenged intradermally with 3 × 104 B16 cells and treated with 1 mg/mouse of DTA-1 Ab or control rat IgG Ab 1, 4, and 9 days after challenge. On day 21, CD4+ and CD8+ T cells purified from splenocytes and lymph nodes of tumor-bearing mice were adoptively transferred at a 2:1 ratio into Rag 1−/− mice. One day after transfer, Rag1−/− recipients were challenged intradermally with 3 × 104 B16 cells. A control group of mice did not receive T cells (No T cells). Mice that received T cells from donors treated (Tx) with control rat IgG Ab are labeled as CD4, CD8, and CD4+CD8 from rat IgG Tx, and mice that received T cells form DTA-1-treated donors are labeled as CD4, CD8, and CD4+CD8 from DTA-1 Tx. Comparisons between all groups that received cells from rat IgG-treated mice (blue lines) vs all groups that received T cell from DTA-1-treated mice (red lines) are statistically significant (p < 0.04).
To demonstrate whether T cells were sufficient for tumor rejection, CD4+ and CD8+ cells purified from spleens and draining lymph nodes of mice challenged with B16 and treated with DTA-1 or control Ab were adoptively transferred into Rag 1−/− recipients (Fig. 3⇑B). One day after transfer, the recipients were challenged with B16 cells. Transfer of CD4+, CD8+ T cells, or both derived from mice treated with control Ab and challenged with B16 did not affect tumor growth. Conversely, transfer of T cells derived from mice treated with DTA-1 and challenged with B16 (CD4+, CD8+, or both) resulted in significant rejection of B16.
To test the therapeutic effect of anti-GITR stimulation, DTA-1 treatment was delayed until mice were bearing tumors of 3-mm mean diameter. Mice were treated with DTA-1 (1 mg/mouse) 10 and 14 days after challenge. None of the mice under these conditions were able to reject the B16 challenge (data not shown). Because B16 is a highly aggressive tumor, it remains to be established whether in slower progressive tumors, anti-GITR demonstrates therapeutic effects.
A number of studies have demonstrated a role for IFN-γ in tumor immunity (31, 32). To test whether IFN-γ was relevant for GITR-mediated tumor rejection, IFN-γ−/− and wild-type mice were treated with DTA-1 and challenged with B16 (Fig. 4⇓). IFN-γ−/− mice treated with DTA-1 were not protected from B16 challenge, demonstrating that this cytokine is required for DTA-1-mediated tumor rejection. We next tested the requirements for perforin and FasL in this system. All pfp−/− mice challenged with B16 and treated with control Ab developed tumors (10 of 10 tumor-bearing mice), whereas most pfp−/− mice treated with DTA-1 showed rejection (2 of 10 tumor-bearing mice). Mice deficient in FasL (gld) challenged with B16 develop tumors (10 of 10 tumor-bearing mice), whereas DTA-1 treatment did not enhance tumor rejection (8 of 10 tumor-bearing mice). We conclude that perforin is not required for GITR-mediated tumor rejection, whereas IFN-γ and FasL are required for B16 rejection.
DTA-1-induced tumor rejection is dependent on IFN-γ and FasL and independent of perforin. A, Wild-type and IFN-γ−/− C57BL/6 mice were treated with control Ab or DTA-1 and challenged intradermally with 3 × 104 B16 cells. All mice received Ab at 1 mg/mouse on days +1, +4, and +9 after challenge. Data are representative of two independent experiments (n = 6–10). B, Wild-type, perforin−/− (pfp−/−), and FasL-deficient (gld) C57BL/6 mice were treated with control Ab or DTA-1 and challenged intradermally with 3 × 104 B16 cells. All mice received Ab at 1 mg/mouse on days +1, +4, and +9 after challenge. Data are representative of two independent experiments (n = 10).
To examine Ag specificity of T cells during tumor rejection, mice were challenged with B16, treated with DTA-1 or control Ab, and analyzed by IFN-γ ELISPOT (Fig. 5⇓, A and B). Mice that received B16 and DTA-1 demonstrated T cell responses against three melanoma differentiation Ags (gp100, TYRP1/gp75, and DCT for CD8+ responses) and against syngeneic B16 cells (for CD4+ and CD8+ responses). Mice that received B16 and control Ab showed no detectable IFN-γ-producing T cells. Interestingly, even though these peptide-specific responses were only detected in the DTA-1 group, low-level responses also existed against negative controls (syngeneic EL4 pulsed with irrelevant peptide or syngeneic splenocytes) (Fig. 5⇓A), suggesting some recognition of self-Ags during early tumor progression. Furthermore, at day 34 after challenge, control animals bearing tumors still remained tolerant/ignorant to these Ags, whereas Ag-specific responses were detected in the DTA-1-treated group that rejected tumor (Fig. 5⇓B).
DTA-1-treated mice that reject B16 recognize melanoma differentiation Ags. T cells from naive mice (naive) or mice challenged with 3 × 104 B16 cells treated with rat IgG control Ab (rat) or DTA-1 (DTA) were purified at day 7 (A) and day 37 (B) and tested in IFN-γ ELISPOT assays against EL4 cells pulsed with peptides or against syngeneic B16 tumor pretreated with Con A supernatant. A, CD4+ T cell (left panel) and CD8+ T cell (right panel) IFN-γ spots at day 7 postchallenge from unchallenged mice (naive; n = 2 mice), mice challenged with B16 and treated with rat IgG control Ab (rat; n = 3), and mice challenged with B16 and treated with DTA-1 (DTA-1; n = 3). B, CD8+ T cell IFN-γ spots at day 37 postchallenge, from unchallenged mice (naive; n = 1), challenged mice treated with rat IgG control Ab (rat; n = 2), and challenged mice treated with DTA-1 (DTA-1; n = 4).
To test whether memory against B16 was established in mice that received DTA-1 and rejected tumor, mice were re-challenged 60–140 days after the primary challenge. Although all naive control mice rapidly developed tumors, only 23% of mice rechallenged with B16 developed tumors (Table I⇓). These results are consistent with the generation of a memory response against B16.
Careful examination of coat color demonstrated that a small proportion of mice (5 of 43 mice) that were treated with DTA-1 and rejected B16, developed signs of mild depigmentation (Table I⇑). Two mice presented mild salt and pepper hair hypopigmentation, whereas the remaining three developed one or two 2- to 3-mm2 patches of hypopigmented hair at the site of tumor challenge. No change in coat color was detected in mice treated with control Ab and challenged with B16. These results indicate that mild autoimmunity was induced in a small subset of DTA-1-treated mice.
To test whether DTA-1 had an effect on augmenting T cell activation, mice were challenged with B16, treated with DTA-1, and sacrificed 7 days after tumor challenge. The analysis of T cell activation markers showed a significant increase in the absolute numbers of CD4+CD44high T cells (Fig. 6⇓A). We gated on CD44 and CD62L (Fig. 6⇓B) to determine the percentage of central memory (CD44high and CD62L+) vs effector T cells (CD44high and CD62L−). There was a significant increase in the percentage of effector T cells in the DTA-1-treated mice compared with control mice (Fig. 6⇓C). We also noted an increased percentage of CD44highCD4+ and CD8+ T cells in draining lymph nodes of the DTA-1-treated group compared with control mice (Fig. 6⇓D).
GITR stimulation increases CD4+ and CD8+ T cell activation. Spleens from C57BL/6 mice intradermally challenged with 3 × 104 B16 cells and treated with a 1 mg/mouse of rat IgG control Ab or DTA-1 at days +1 and +4 were harvested at day 7 and analyzed by flow cytometry. A, Splenic T cells gated on CD3+, CD4+, or CD8+, and CD44high. B, Analysis of T cells gated on CD3+, CD4+, or CD8+, comparing CD44 vs CD62L expression. C, Percentage of T cells gated on CD44−CD62L+ (black), CD44−CD62L− (light gray), CD44highCD62L− (dark gray), and CD44highCD62L+ (white) in the right panel. D, Inguinal draining lymph nodes (DLN) and nondraining lymph nodes (NDLN) from animals treated with rat IgG control Ab or DTA-1 were harvested 7 days after tumor challenge, pooled, and analyzed by flow cytometry. Data are representative of two independent experiments (n = 4 mice per group). ∗, p < 0.05.
Previous studies have demonstrated that GITR-stimulated Treg proliferate in the presence of IL-2 (11, 12, 13, 16, 33). We repeated these experiments and found that, indeed, Treg in the presence of CD3 stimulation and IL-2 readily expand, and proliferation is increased in the presence of CD28 or DTA-1 (data not shown), confirming its costimulatory role on Treg. We next tested whether systemic administration of DTA-1 to naive hosts could expand the absolute number of CD4+CD25+ T cells. In both C57BL/6 and BALB/c mice, weekly treatments with 1 mg/mouse of DTA-1 increased the absolute number of CD4+CD25+ T cells compared with mice treated with control Ab (data not shown).
Because in vivo GITR stimulation expands Treg, we tested whether once Treg had been stimulated through GITR, the expanded cells could still suppress effector T cells. High precursor frequencies and strong proliferation is observed during allogeneic responses, thus we used this model to test suppression (Fig. 7⇓). Treg were purified and cultured with anti-CD3 and anti-GITR for a week and subsequently tested in a MLR (Fig. 7⇓A). GITR-stimulated cultured Treg were able to suppress alloactivation of CD4+CD25− and CD8+CD25− to a better extent than fresh Treg. Cultured Treg are better suppressors than freshly isolated Treg, presumably due to the fact that all GITR-cultured Treg added to the MLR had been polyclonally stimulated with anti-CD3. Next, we tested whether Treg isolated from mice treated with DTA-1 maintained a suppressive activity ex vivo (Fig. 7⇓B). Naive mice were treated with 1 mg/mouse of a control Ab (rat IgG) or with DTA-1. Twenty-four hours after treatment, Treg were isolated and tested in a MLR. Treg derived from DTA-1-treated mice were equally efficient at suppressing alloactivation than Treg derived from the control group. These results suggest that once GITR stimulation has occurred, Treg regain their suppressive activity.
Prior GITR stimulation does not impair Treg suppression. A, Treg cultured in vitro under GITR stimulation. Sorted C57BL/6 CD4+CD25+ T cells (postsort purity >98%) cultured in vitro for 7 days in the presence of anti-CD3 (1 μg/ml), DTA-1 (10 μg/ml), and IL-2 (100 U/ml) (Cult. Treg) or freshly sorted CD4+CD25+ T cells (Fresh Treg) were added to a MLR at a 1:1 ratio with responding T cells. MLR consisted of responding C57BL/6 CD4+CD25− or CD8+CD25− T cells (1 × 104/well) stimulated with irradiated BALB/c splenocytes (2 × 104/well) (Stimulators). B, Treg isolated from DTA-1-treated mice. Sorted CD4+CD25+ T cells (postsort purity >98%) from naive C57BL/6 mice treated (Tx) with 1 mg/mouse of DTA-1 Ab (Treg from DTA-1 Tx) or control Ab (Treg from rat IgG Tx) 24 h before Treg purification were added to MLR at a 1:1 ratio with responding T cells. MLR consisted of responding C57BL/6 whole T cells (T cells) (1 × 104/well) stimulated with irradiated BALB/c splenocytes (2 × 104/well) (Stimulators). The data represent thymidine incorporation for 18 h after 3 days of culture. ∗, p < 0.05.
To test the role of DTA-1 administration on Treg expansion in tumor-bearing mice, T cells from spleens and lymph nodes of mice challenged with B16, treated with DTA-1, and sacrificed 12 days after tumor challenge were analyzed by flow cytometry (Fig. 8⇓). It has been demonstrated that FoxP3 is a required and restricted marker for natural Treg (34, 35). Mice treated with DTA-1 have increased percentage and absolute number of splenic FoxP3+ cells. The percentage of FoxP3+ in lymph node from these mice compared with the control group was similar, but the absolute number of these cells was increased in the DTA-1-treated group (data not shown). These results demonstrate an expansion of FoxP3+ cells after DTA-1 treatment and are consistent with previous studies demonstrating that GITR stimulation induces Treg proliferation in vitro (11, 12, 13, 16, 33).
GITR stimulation induces the expansion of CD4+FoxP3+ cells in tumor-bearing mice. Mice were challenged intradermally with 3 × 104 B16 cells and treated with either rat IgG control Ab or DTA-1 Ab after 1, 4, and 9 days from tumor challenge. Mice were sacrificed 12 days after tumor challenge, and splenocytes were analyzed by flow cytometry. A, Representative flow cytometry analysis of splenocytes including an isotype control (Control), treatment with control Ab treatment (rat IgG), and treatment with anti-GITR agonist Ab (DTA-1). Percentage (B) and absolute number (C) of CD4+FoxP3+ cells in spleens. Data are representative of two independent experiments (n = 4 mice per group). ∗, p < 0.05.
Our results indicate that GITR stimulation overcomes tolerance/ignorance to self-Ags (melanoma differentiation Ags), induces tumor rejection that requires T and NK/NKT cells, and is dependent on IFN-γ and FasL, but independent of perforin. In vivo GITR stimulation induces potent rejection and memory against B16, whereas it induces mild hypopigmentation and expansion of FoxP3+ cells.
We sought to explore the mechanism of B16 tumor rejection. Adoptive transfer of T cells derived from DTA-1-treated donor demonstrates that CD4+ and CD8+ T cells are sufficient for GITR-mediated rejection of B16. Although most studies implicate CD8+ T cells at the effector arm of tumor rejection, as our results also demonstrate, it was unexpected to identify B16 rejection mediated by CD4+ T cells derived from DTA-1-treated mice. Studies have shown that CD4+ T cells that infiltrate tumors contain perforin granules and have the potential to kill by Fas/FasL system (36, 37). Because class II Ags are expressed in different solid malignancies including melanoma (38, 39), CD4+ T cells may target these tumors directly and secrete IFN-γ to activate macrophages that reject the tumor. Alternatively, in antitumor responses against class II-negative tumors, the melanoma tumors may be cross-presented by host APC that activate CD4+ T cells. Once activated in the lymph node, CD4+ T cells migrate back to the tumor, where they become activated by macrophages that present class II-restricted tumor Ags. Once locally reactivated, CD4+ T cells generate IFN-γ-activating macrophages and NK for tumor lysis (40). A similar IFN-γ-dependent mechanism may operate in GITR-mediated CD4+ rejection of B16. Although IFN-γ up-regulates the expression of class I and induces the expression of class II on B16 cells cultured in vitro, expression of class I and class II in B16 cells harvested from tumor-bearing mice is heterogeneous for both molecules (data not shown). Because the recipients of the immunized T cells were Rag 1−/− mice, it is possible that tumor-specific CD4+ T cells migrate to the tumor, become reactivated, secrete IFN-γ, target the tumor directly, and/or activate macrophages and NK cells that eradicate B16. Importantly, because the purified cells used in the adoptive transfer were not depleted of CD25+ cells before transfer into Rag 1−/− recipients, the CD4+ immune cells contained normal numbers of Treg. These mice rejected B16, indicating that CD4+ effector cells can induce tumor rejection even in the presence of Treg. These results are consistent with a previous study in which three mice that were adoptively transferred with CD4+ T cells from donors immunized with B16 and treated with anti-CD25, rejected tumors (3). Because our results indicate that IFN-γ and FasL are required for DTA-1-mediated B16 rejection, these cytolytic mechanisms, especially FasL, may be involved in CD4+ rejection of B16. Future adoptive transfer experiments using CD4+ T cells deficient in FasL or other effector molecules will elucidate the pathways involved in CD4+ DTA-1-mediated tumor rejection.
Our data indicate an important role for IFN-γ in B16 rejection, consistent with its role in immunosurveillance and tumor rejection (31). Although IFN-γ produced by CD8+ T cells responding to CTL epitopes inhibit the generation/activation of CD4+CD25+ Tregs (41), recent studies have indicated that IFN-γ production by Treg is required for suppression (42, 43, 44). Our results demonstrate that the predominant role for IFN-γ in GITR-dependent tumor rejection is on effector T cells, because GITR stimulation does not protect IFN-γ−/− hosts from B16 challenge.
Finally, we hypothesize that priming against tumors occurs and is strongly enhanced by GITR stimulation. We believe that GITR stimulation may enhance antitumor immunity through a very potent costimulatory signal on effector cells, which allows for its remarkable activity against nonimmunogenic aggressive tumors such as B16. This strong costimulation of effector T cells allows for long-term memory to be generated. Although other groups have determined that GITR stimulation inhibits Treg suppression (9, 10, 11, 12), our experiments do not demonstrate that GITR stimulation abrogates Treg suppression, but clearly demonstrate that GITR costimulation of effector T cells allows for rejection of the aggressive B16 melanoma line. Future experiments with T cells deficient in GITR (GITR−/−) will demonstrate whether tolerance/ignorance is broken through a strong costimulatory signal provided by GITR stimulation to the effector T cells or whether, as other groups have determined, it is due to GITR stimulation on Treg that abrogates suppression.
Overall, our results in preclinical mouse models for cancer rejection indicate that systemic GITR stimulation breaks self-tolerance/ignorance, allows for recognition of tumor Ags, and induces tumor rejection. GITR stimulation is a potent and promising strategy to enhance tumor immunotherapy without the detrimental effect of autoimmunity.
T. Ramirez-Montagut thanks Humilidad Gallardo and Nathalie E. Blachere for helpful comments and Robert B. Darnell for his support in the elaboration of this manuscript.
↵1 This work was supported by Grants HL69929, HL72412, and CA107096 from the National Institutes of Health, Awards from the Emerald Foundation and The Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center funded by William H. Goodwin and Alice Goodwin, the Commonwealth Foundation for Cancer Research (to M.R.M.v.d.B.), National Cancer Institute Grants CA33049, CA56821, CA47179, CA59350, and Swim Across America (to A.N.H.).
↵3 Abbreviations used in this paper: Treg, regulatory T cell; TNFR, TNF receptor; GITR, glucocorticoid-induced TNFR family related gene; FasL, Fas ligand.
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McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. Collins, M. C. Byrne. 2002. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16: 311-323.
Ronchetti, S., O. Zollo, S. Bruscoli, M. Agostini, R. Bianchini, G. Nocentini, E. Ayroldi, C. Riccardi. 2004. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur. J. Immunol. 34: 613-622.
Ji, H. B., G. Liao, W. A. Faubion, A. C. Abadia-Molina, C. Cozzo, F. S. Laroux, A. Caton, C. Terhorst. 2004. Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J. Immunol. 172: 5823-5827.
Muriglan, S. J., T. Ramirez-Montagut, O. Alpdogan, T. W. Van Huystee, J. M. Eng, V. M. Hubbard, A. A. Kochman, K. H. Tjoe, C. Riccardi, P. P. Pandolfi, et al 2004. GITR activation induces an opposite effect on alloreactive CD4+ and CD8+ T cells in graft-versus-host disease. J. Exp. Med. 200: 149-157.
Valzasina, B., C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, M. P. Colombo. 2005. Triggering of OX40 (CD134) on CD4+CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR. Blood 105: 2845-2851.
Kanamaru, F., P. Youngnak, M. Hashiguchi, T. Nishioka, T. Takahashi, S. Sakaguchi, I. Ishikawa, M. Azuma. 2004. Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J. Immunol. 172: 7306-7314.
Kohm, A. P., J. S. Williams, S. D. Miller. 2004. Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis. J. Immunol. 172: 4686-4690.
Tone, M., Y. Tone, E. Adams, S. F. Yates, M. R. Frewin, S. P. Cobbold, H. Waldmann. 2003. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc. Natl. Acad. Sci. USA 100: 15059-15064.
Turk, M. J., J. A. Guevara-Patino, G. A. Rizzuto, M. E. Engelhorn, S. Sakaguchi, A. N. Houghton. 2004. Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J. Exp. Med. 200: 771-782.
Calmels, B., S. Paul, N. Futin, C. Ledoux, F. Stoeckel, B. Acres. 2005. Bypassing tumor-associated immune suppression with recombinant adenovirus constructs expressing membrane bound or secreted GITR-L. Cancer Gene. Ther. 12: 198-205.
Fidler, I. J., G. L. Nicolson. 1978. Tumor cell and host properties affecting the implantation and survival of blood-borne metastatic variants of B16 melanoma. Isr. J. Med. Sci. 14: 38-50.
Poste, G., J. Doll, I. J. Fidler. 1981. Interactions among clonal subpopulations affect stability of the metastatic phenotype in polyclonal populations of B16 melanoma cells. Proc. Natl. Acad. Sci. USA 78: 6226-6230.
Ponomarev, V., M. Doubrovin, I. Serganova, J. Vider, A. Shavrin, T. Beresten, A. Ivanova, L. Ageyeva, V. Tourkova, J. Balatoni, et al 2004. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur. J. Nucl. Med. Mol. Imaging 31: 740-751.
Overwijk, W. W., A. Tsung, K. R. Irvine, M. R. Parkhurst, T. J. Goletz, K. Tsung, M. W. Carroll, C. Liu, B. Moss, S. A. Rosenberg, N. P. Restifo. 1998. gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand. J. Exp. Med. 188: 277-286.
Seaman, W. E., M. Sleisenger, E. Eriksson, G. C. Koo. 1987. Depletion of natural killer cells in mice by monoclonal antibody to NK-1.1: reduction in host defense against malignancy without loss of cellular or humoral immunity. J. Immunol. 138: 4539-4544.
Kurosawa, S., M. Harada, G. Matsuzaki, Y. Shinomiya, H. Terao, N. Kobayashi, K. Nomoto. 1995. Early-appearing tumour-infiltrating natural killer cells play a crucial role in the generation of anti-tumour T lymphocytes. Immunology 85: 338-346.
Leist, T. P., M. Kohler, M. Eppler, R. M. Zinkernagel. 1989. Effects of treatment with IL-2 receptor specific monoclonal antibody in mice. Inhibition of cytotoxic T cell responses but not of T help. J. Immunol. 143: 628-632.
Moreau, J. L., M. Nabholz, T. Diamantstein, T. Malek, E. Shevach, J. Theze. 1987. Monoclonal antibodies identify three epitope clusters on the mouse p55 subunit of the interleukin 2 receptor: relationship to the interleukin 2-binding site. Eur. J. Immunol. 17: 929-935.
Lowenthal, J. W., P. Corthesy, C. Tougne, R. Lees, H. R. MacDonald, M. Nabholz. 1985. High and low affinity IL 2 receptors: analysis by IL 2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J. Immunol. 135: 3988-3994.
Ikeda, H., L. J. Old, R. D. Schreiber. 2002. The roles of IFN γ in protection against tumor development and cancer immunoediting. Cytokine Growth Factor Rev. 13: 95-109.
Stephens, G. L., R. S. McHugh, M. J. Whitters, D. A. Young, D. Luxenberg, B. M. Carreno, M. Collins, E. M. Shevach. 2004. Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J. Immunol. 173: 5008-5020.
Hishii, M., J. T. Kurnick, T. Ramirez-Montagut, F. Pandolfi. 1999. Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes. Clin. Exp. Immunol. 116: 388-394.
Homma, S., H. Komita, Y. Sagawa, T. Ohno, G. Toda. 2005. Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12. Immunology 115: 451-461.
Altomonte, M., E. Fonsatti, A. Visintin, M. Maio. 2003. Targeted therapy of solid malignancies via HLA class II antigens: a new biotherapeutic approach?. Oncogene 22: 6564-6569.
Albino, A. P., A. N. Houghton, M. Eisinger, J. S. Lee, R. R. Kantor, A. I. Oliff, L. J. Old. 1986. Class II histocompatibility antigen expression in human melanocytes transformed by Harvey murine sarcoma virus (Ha-MSV) and Kirsten MSV retroviruses. J. Exp. Med. 164: 1710-1722.
Corthay, A., D. K. Skovseth, K. U. Lundin, E. Rosjo, H. Omholt, P. O. Hofgaard, G. Haraldsen, B. Bogen. 2005. Primary antitumor immune response mediated by CD4+ T cells. Immunity 22: 371-383.
Nishikawa, H., T. Kato, I. Tawara, H. Ikeda, K. Kuribayashi, P. M. Allen, R. D. Schreiber, L. J. Old, H. Shiku. 2005. IFN-γ controls the generation/activation of CD4+CD25+ regulatory T cells in antitumor immune response. J. Immunol. 175: 4433-4440.
Sawitzki, B., C. I. Kingsley, V. Oliveira, M. Karim, M. Herber, K. J. Wood. 2005. IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J. Exp. Med. 201: 1925-1935.
Kelchtermans, H., B. De Klerck, T. Mitera, M. Van Balen, D. Bullens, A. Billiau, G. Leclercq, P. Matthys. 2005. Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ. Arthritis Res. Ther. 7: R402-R415.
Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, M. L. Alegre, P. Puccetti. 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212.


The campsite Camping Belmare is located in the midst of a beautiful pine forest, on a stretch of the coast of Marina di Castagneto. The Mediterranean vegetation - full of oaks, tamarisks, junipers and pines - protects the camping area from the sun by creating shade: perfect for relaxation and hot summer days. A sandy beach awaits all sun lovers, who can choose whether or not to take advantage of the services provided while enjoying a beautiful day at the sea. The structure is strategically located near the major Tuscan art cities such as Florence, Pisa, Siena and San Gimignano, which can easily be reached by car. Furthermore, the harbor of Piombino is also in the vicinity. From Piombino, one can take a mandatory trip to visit the Isle of Elba during his stay.
The campsite Camping Belmare is ideal for families and small groups and it is suitable for both a short or long holiday. The Camping offers a series of services such as a self-service-bar and pizzeria, a newspaper shop and a fruits and vegetables store. Furthermore, it is also very close to the town of Marina di Castagneto, which allows one to take advantage of all of the services that are provided outside the camp area. The camp welcomes caravans, campers and tents, but it also has bungalows available so that one is free to add a touch of comfort to the adventurous camp life.


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text:
Q: dynamic popup window mvvm light wpf I have a problem with my mvvm project:
In my project i have a listview that contains a list of different kinds of objects,
and i want to make a pop up window that will show additional info about each object.
The popup shows up when you click on a item in the listView..
I have two problems with this,

*

*how do i send the selected item from the view model of the list to the popup?

*how do i dynamically change the style of the content inside the popup by the type of item that was sent to it.

Thanks...

A: You should bind the DataContext property of the popup to the selected item property of the viewmodel.
Also, in the popup definition, you can use DataTemplates, and its DataType property. In the popup resources, you can define a DataTemplate for each type you want to display, and configure the DataType to this type. In the content of the popup, a simple ContentControl with the Content bound to the DataContext itself will to the trick.
Hope it answered your problem.

redpajama_set_name: RedPajamaStackExchange


DAD is a complete audio playout automation and control system for radio and television.
DAD's incredibly rich feature set enables television and radio broadcasters to ensure reliable, accurate delivery of all audio content on time, every time.
USER FRIENDLY: Award winning Presenter interface for live assist Playout. Fast paced changes easily made on the fly. Customizable module based architecture.
POWERFUL ENGINE: Powerful automation engine allows for up to 16 separate playlists to be run from one workstation simultaneously.
SCALABLE DESIGN: Whether you need a single workstation or 100, a DAD system can be designed to fit you now and into the future.
PERFORMANCE ORIENTED: DAD is built for speed and reliability. High-speed database allows for instant searching and sorting. Scrollable lists allow you to view all of your assets instantaneously.
CENTRALIZED CONTENT: Share content across multiple stations across multiple locations. Easily break away for local commercials, liners and jingles and rejoin seamlessly.
EASY INTEGRATION: Seamlessly integrate into any environment. DAD interfaces with all kinds of broadcast hardware and software.
SUPPORTS ANY LANGUAGE: Unicode compliant. DAD will work with any character set in any language.
AUTOMATIC PLAYLIST CREATION: Automatically create playlists out of music and commercial logs. Re-export traffic and without having to re-merge the logs.
FLEXIBLE COMMANDS: Integral programming language allows for construction of commands and macros to operate the system more efficiently.
SCHEDULING: Integrated scheduling software plus tight bi-directional interface to leading scheduling systems.
REAL-TIME LOG: Real-time log changes in any studio at any time. No need to reload the current hour or current day's log.
DAD is a complete playout solution scalable from the largest multinational television or radio broadcast application to, online streaming and web casters or small closed-circuit systems found in education, retail and hospitality.
DAD is a third generation software system built upon ENCO's proven and widely accepted family of products in use at thousands of facilities worldwide.
Running on standard off-the-shelf PC hardware, DAD's complete toolkit allows broadcasters to implement an audio delivery and automation system that precisely meets their needs. Combining rich functionality of multiple reproducers or "players" and recorders, flexible array panels, and user-defined hot keys, DAD enables broadcasters to optimize performance for their specific requirements. It provides a platform for integrating the many sources of content flowing into today's modern broadcast environment including news, traffic, weather, and scheduling, as well as reconciliation to billing and corporate information systems.
DAD is available as software only, or as a turnkey system delivered with ENCO's tested and proven hardware configurations.
Want to know more about the software? contact us today, or download the PDF and take a read at your leisure.


text:
Phycella é um género botânico pertencente à família Amaryllidaceae.

Espécies
 Phycella australis Ravenna, Pl. Life 37: 71 (1981).
 Phycella brevituba Herb., Amaryllidaceae: 154 (1837).
 Phycella cyrtanthoides (Sims) Lindl., Bot. Reg. 11: t. 928 (1825).
 Phycella herbertiana Lindl., Edwards's Bot. Reg. 16: t. 1341 (1830).
 Phycella scarlatina Ravenna, Pl. Life 37: 69 (1981).

 Plant systematics
 PPP-index
 The International Plant Names Index
 Catalogue of Life
 Encyclopedia of Life

Referências

Amaryllidaceae
Géneros de plantas

redpajama_set_name: RedPajamaWikipedia


text:
LON:GNK
Greene King Share Forecast, Price & News
Adding Greene King plc
GBX 849.20
Receive GNK News and Ratings via Email
Sign-up to receive the latest news and ratings for Greene King and its competitors with MarketBeat's FREE daily newsletter.
About Greene King
Greene King plc operates as a pub retailer and brewer in the United Kingdom. The company operates through three segments: Pub Company, Pub Partners, and Brewing & Brands. Its brands include Greene King Local Pubs, Hungry Horse, Flaming Grill, Farmhouse Inns, and Chef & Brewer. The company is also involved in brewing, marketing, and selling beer under the Greene King IPA, Old Speckled Hen, Abbot Ale, and Belhaven Best brands. In addition, Greene King plc engages in the employment, financing, pension trustee, and property businesses. As of April 30, 2018, it operated 2,855 managed, tenanted, leased, and franchised pubs, restaurants, and hotels. Greene King plc was founded in 1799 and is headquartered in Bury St Edmunds, the United Kingdom.
Greene King (LON:GNK) Stock Crosses Above 200 Day Moving Average of $849.20
January 12, 2022 | americanbankingnews.com
March 29, 2021 | reuters.com
(LSE:SPRT)
January 10, 2021 | fool.co.uk
Greene King Acquisition By CK Asset Gains European Commission Approval
October 17, 2019 | www.lse.co.uk
EU clears Greene King acquisition
Greene King Reports European Commission Clearance For Cash Offer
October 17, 2019 | www.finanznachrichten.de
Capital Group Cuts Whilst JPMorgan Builds Greene King Stake
September 27, 2019 | www.lse.co.uk
Greene King Faces Shareholder Opposition To Remuneration Report
September 6, 2019 | www.morningstar.co.uk
What to Watch: German recession fears, Berkeley soars and Greene King sales fall
September 6, 2019 | ca.finance.yahoo.com
Greene King 18-week LFL Sales In Pub Company Down
September 6, 2019 | www.finanznachrichten.de
Greene King Regulatory News
September 5, 2019 | www.lse.co.uk
The death of Britain's pubs has been greatly exaggerated
August 26, 2019 | finance.yahoo.com
StockBeat: The Great Brexit Garage Sale is on as Hasbro Snaps up Peppa Pig
www.greeneking.co.uk
GBX 59.81 per share
GBX 680.20 per share
There is not enough analysis data for Greene King.
Greene King has received 688 "outperform" votes. (Add your "outperform" vote.)
Greene King has received 168 "underperform" votes. (Add your "underperform" vote.)
Greene King has received 80.37% "outperform" votes from our community.
MarketBeat's community ratings are surveys of what our community members think about Greene King and other stocks. Vote "Outperform" if you believe GNK will outperform the S&P 500 over the long term. Vote "Underperform" if you believe GNK will underperform the S&P 500 over the long term. You may vote once every thirty days.
Greene King is a leading dividend payer. It pays a dividend yield of 3.91%, putting its dividend yield in the top 25% of dividend-paying stocks.
Greene King does not have a long track record of dividend growth.
The dividend payout ratio of Greene King is 0.85%. This payout ratio is at a healthy, sustainable level, below 75%.
In the past three months, Greene King insiders have not sold or bought any company stock.
The P/E ratio of Greene King is 21.94, which means that it is trading at a more expensive P/E ratio than the market average P/E ratio of about 14.90.
The P/E ratio of Greene King is 21.94, which means that it is trading at a less expensive P/E ratio than the Services sector average P/E ratio of about 37.70.
Greene King has a P/B Ratio of 1.25. P/B Ratios below 3 indicates that a company is reasonably valued with respect to its assets and liabilities.
Greene King (LON:GNK) Frequently Asked Questions
How were Greene King's earnings last quarter?
Greene King plc (LON:GNK) issued its quarterly earnings data on Thursday, November, 29th. The company reported $33.10 EPS for the quarter.
View Greene King's earnings history.
Is Greene King a good dividend stock?
Greene King pays an annual dividend of GBX 0.33 per share and currently has a dividend yield of 3.91%. The dividend payout ratio of Greene King is 0.85%. This payout ratio is at a healthy, sustainable level, below 75%.
View Greene King's dividend history.
Who are Greene King's key executives?
Greene King's management team includes the following people:
Mr. Rooney Anand, CEO & Exec. Director (Age 55)
Mr. Richard Smothers, CFO & Director (Age 51)
Mr. Richard Lewis, Chief Operating Officer (Age 48)
Mr. Mark Blythman, IR Director
Mr. Andrew Bush, Group HR Director
Who are some of Greene King's key competitors?
Some companies that are related to Greene King include HelloFresh (HFG), Whitbread (WTB), SSP Group (SSPG), Domino's Pizza Group (DOM), Cara Operations (CARA), Mitchells & Butlers (MAB), MTY Food Group (MTY), Ei Group plc (EIG.L) (EIG), J D Wetherspoon (JDW), Young & Co.'s Brewery, P.L.C. (YNGA), The Restaurant Group (RTN), A and W Revenue Royalties Income Fund (AW.UN), Marston's (MARS), Young & Co.'s Brewery, P.L.C. (YNGN) and Fuller, Smith & Turner (FSTA).
View all of GNK's competitors.
What other stocks do shareholders of Greene King own?
Based on aggregate information from My MarketBeat watchlists, some companies that other Greene King investors own include Coats Group (COA), Barratt Developments (BDEV), Old Mutual (OML), Tesco (TSCO), GREENE KING PLC/S (GKNGY), Hurricane Energy (HUR), Kier Group (KIE), Link Motion (LKM), National Grid (NG) and Universal Display (OLED).
What is Greene King's stock symbol?
Greene King trades on the London Stock Exchange (LON) under the ticker symbol "GNK."
How do I buy shares of Greene King?
Shares of GNK and other U.K. stocks can be purchased through online brokerage accounts that support trading on the London Stock Exchange (LSX). Some U.K. companies can be purchased through major U.S. brokerages in the form of American Depository Receipts (ADRs), which are placeholder equities held in a trust by a bank that represent shares of a foreign stock.
What is Greene King's stock price today?
One share of GNK stock can currently be purchased for approximately GBX 849.20.
How much money does Greene King make?
Greene King has a market capitalization of £2.63 billion and generates £2.22 billion in revenue each year.
How many employees does Greene King have?
Greene King employs 39,000 workers across the globe.
What is Greene King's official website?
The official website for Greene King is www.greeneking.co.uk.
Where are Greene King's headquarters?
Greene King is headquartered at Abbot House, Westgate Street, BURY ST. EDMUNDS, IP33 1QT, United Kingdom.
How can I contact Greene King?
Greene King's mailing address is Abbot House, Westgate Street, BURY ST. EDMUNDS, IP33 1QT, United Kingdom. The company can be reached via phone at +44-1284-763222.


This American Expedition Stoneware Coffee Mug features a full-wrap illustrated design of a whitetail deer and the phrase "Deer Camp - Huge Racks and Sketchy Facts." This attractive, large mug has a capacity of 15 ounces and a large handle. The mug is first glazed with a "bone" colored outside and black inside. This mug is the perfect gift for the deer hunter or sportsman in your life.


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Halloween is a time for yummy treats and great fun. It's also a time to get creative with baking, so think of some of your favourite recipes and how you can add a Halloween twist to it. It's pretty easy to turn Rice Krispie Squares into any holiday treat simply by adding food colouring or sprinkles.
If you can find thicker pretzels, it'll probably look better for the stem of the pumpkin. Also, if you can find a tube of icing to decorate the face, you'll be able to make actual Jack-O-Lantern faces, as oppose to my toothpick drawn smiley faces.
In a large pot melt the butter on low heat. Once it has melted add in the marshmallows.
Continue to stir the marshmallow until melted and add in the vanilla and food colouring. If you use red and yellow, drop in the yellow first and then the red, as it is harder to make the marshmallow lighter than darker. Add in about 5 drops of yellow and 2 drops of red. Add in either more red or yellow to get your desired colour of orange.
Once you have blended the colour into the marshmallow add the Rice Krispie and mix quickly.
Butter your hands so you don't get the mixture stuck to your hands and roll the mixture into a ball. Take your index knuckle and make a dent on the top where you will stick the pretzel as the stem.
Decorate the faces on the little pumpkins and enjoy!
Last weekend I went to Banff for the Thanksgiving long weekend. It's always such a nice break for me to head out west to the Rocky Mountains as Toronto can get very hectic sometimes.
Usually when you think of restaurants in Banff, you think of really great pubs where you can grab a beer with friends. However, tucked away in the Sundance Mall is this amazing vegetarian bistro called Nourish.
You would be surprised by the food that comes out of the small little kitchen, the restaurant itself, can't really hold more than 30 people. I can assure you that you will experience many unique and sweet flavours at this restaurant. I didn't even notice that I was just eating vegetables, the absence of meat did not cross my mind once, that is how flavourful the food is here.
The food is also meant to be shared, which is a wonderful idea considering you want to try everything on the menu. There was five of us so we ordered 4 dishes and 2 appetizers to start. It was the perfect amount, I was so content in the end, it even left enough room for their strawberry and apple pie.
The first thing that arrived on the table were our two appetizers. We ordered the Edamame Beans and Spanakopita. These appetizers instantly gave me an idea of what I was in for at this restaurant. New flavours added to old dishes. The Edamame beans were tossed in Hemp Seed Gomasio and drizzled with a creamy sweet sauce. The spanakopitas were filled with 4 spinach and feta, served on a bed of fresh greens with a balkan style yogurt and dill.
We then had the Gourmet Falafel, which contained spiced oven baked chickpeas and fava bean falafael. It was wrapped in a garlic hummus tortilla shell with cucumbers, tomatoes, red onion, roasted red peppers, field greens and garlic dill. It even came with a edible flower, which kinda tasted a bit like lettuce.
We then moved onto their delicous House Curry which contained lentils, spinach and chickpeas stewed in tomato masala with seasoned quinoa and veggies served on rice.
The King Kong Noodle was probably the most flavourful dish of the night. The soup is made up of 6 nuts and veggies in a sweet coconut chili sauce that is topped with cucumbers, spinach, marinated mandarin oranges and pea shoots served with verm rice noodles. Seriously, flavour, flavour, flavour.
Finally, we received our Spicy Salsa Verde Burrito filled with chinnamon chili spiced cauliflour, mushrooms, red pepper, jalapeno, refriend black beans, sticky rice and monterey jack, topped with their salsa verde.
Even though I was completely full at the end I had to try their strawberry and apple pie which made the whole entire meal and night come together at the end.
If you are ever in Banff, I highly recommend you come here with a few friends and enjoy a night of fresh, healthy and flavourful food.
If you're missing out on your mom's Thanksgiving dinner this weekend you should definitely make this dish. It's easy, tasty and it'll feel like a home cooked meal. This is also my friend Cari's favourite dish.
Cut up the chicken breast into bite sized cubes and place them in a 9x1 3" oven dish.
In a large bowl mix together the cranberry sauce, french dressing, onion soup mix and the frozen cranberries. Pour the mixture over the chicken in the oven dish.
Cover the dish with tin foil and bake for 45 minutes, uncovering for the last 10 minutes.
Serve over rice and with a mixed green salad.
I know this fall weather probably isn't making you think about ice cream, but it should. It's never too cold to have Delight's handmade organic ice cream. Greg's Ice Cream at Bloor and Spadina, and White Squirrel in Trinity Bell Woods, serve really great ice cream. But I am seriously telling you, that you have never tried real ice cream, until you've tried it at Delight.
There's something to be said about people who incorporate love and passion into their food, it taste different. Owner's Jennifer Rashleigh and Jeff Brown are a pair that truly care about how their chocolate and ice cream will taste.
Delight uses only natural, organic ingredients, fair trade chocolate and sugar, as well as seasonal and local fruits. These people know how to make ice cream and chocolate, they've stripped it down to the bare essential ingredients.
Two flavours that you must try: Mint Chocolate Chip and their Quebec Blue Cheese. Blue Cheese you say? Yes, Blue Cheese and ice cream, you would have never guessed that they would taste amazing together.


text:
Q: What does the word "there" refer to? He went to the nearest village and (
there) in the bazaar, he found various kinds of meat and fish.

A: It means: in, at, or to that place or position.
When "there" follows a location, it refers to that location. Example:
We went to London and stayed there ten days.
Your sentence can be written to:
He went to the nearest village and (in that village) in the bazaar, he found various kinds of meat and fish.