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CURATORIAL TEAM 2017
PHOTO ARCHIVE 2017
THEORY PROGRAM 2016
about asfaBBQ
The Athens School of Fine Arts [ASFA] barbeque [BBQ] is an international meeting for practitioners, theorists, students and audiences of time-based arts organized by the Theater of ASFA and the Unit of Entrepreneurship and Innovation of ASFA (MoKE).
The program of the asfaBBQ is eclectic as it is international, it is inspired by an experimental and restless attitude that tries to renegotiate our relationship through our bodies. A series of performances, workshops and scores of parallel events that take place over ten days in the buildings of the Athens School of Fine Arts, hopes to produce a new society of pleasure, creativity and emancipation.
asfaBBQ Team
Vassilis Vlastaras Artistic Director of asfaBBQ. He is a visual artist and Associate Professor at the Athens School of Fine Arts. Lives and works in Athens, Greece. He creates large-scale site-specific installations and he experiments with various media in painting, drawing, photography, video, real time video and sound performances and computer interactive installations. His artistic practice examines the relationship between artwork and urban space, the notion of the ephemeral, and the interrelation between spatial movement, material and sound. Currently his work investigates the personal and social implications of loss, oblivion, history, memory and the subsequent acts of remembrance/ memorialization and the relationships between the appetite for life and the frenzy of death. He has taken part in many exhibition projects, solo and group shows in Greece and abroad.
Georgios Papadopoulos PhD, producer of asfaBBQ. He combines economics and philosophical analysis with an exploratory artistic practice. His research gravitates around money and its socioeconomic functions. Between 2008 and 2009 he was Researcher at the Theory Department of the Jan Van Eyck Academy in Maastricht and in 2012 he received the Vilém Flusser Award for Artistic Research by the transmediale festival and the University of the Arts (UdK) in Berlin. He has published many articles on Economics, Artistic Research, and Philosophy and two books; Notes Towards A Critique of Money, 2011 and Grexit, 2012.
Technical administration / Technical support
Thomas Economacos
Kostas Manousis
Curetorial Team 2018
FYTA (meaning: plants) is a conceptual performance duo aiming at the redefinition of the 'radical' in art, as well as the wiping-out of notions of greek tradition and greekness. FYTA initially started operating as a musical duo, a "band", producing their own brand of dyslexic messthetics. In two years, they released 5 (non-)music records, conceptually revolving around metaphors on nature, the 'natural' and naturalised social relations, as well as sabotaging distinctions between the traditional and the modern, exposing the self-importance of the avantgardist and mocking the white-male concept of the precious outsider artist. In their last LP, "Floweremedies", they produced 50 tracks for 50 persons, based on a pseudo/mildly-Lacanian application that the audience had to fill in. The 50 persons varied from friends to total strangers to Greek TV celebrities. At some point they moved on to making videos, having produced "FYTA go to the british museum", a parody of greek nationalist Elgin marbles videos, as well as the documentary form, and caused an uproar with their video "the genes of stupidity", in which a London laboratory claims to have found a genetic malfunction in Greek DNA. The video received 10.000 views in three days, a huge amount of complaints and the threat to sue, as well as two major TV stations requesting permission to show it. It was eventually removed when the person portrayed in it was physically threatened. Alongside their own work, FYTA are active on curation and facilitation. In October 2013, they organized a mammoth 7-day-long, 50-artist participating situationist burlesque at the Athens Biennale. Within this Bible-inspired holy week, FYTA presented work individually or in collaboration with others, in various formations, and curated others' works on the dystopia of contemporary Greece. The centrepiece of the event was the artwork "The 100 worst Greeks" in which the audience had the chance to vote for their own worst league tables, as well as react for/against some of the edgier inclusions. Finally, in 2014, FYTA started their own record label, FYTINI, for the destablisation of Greek music cliches and creating a space for those alienated and marginalised not just by a mainstream, but by an equally suffocating - and more hypocritical - 'alternative culture' scene.
The Ministry of Post-Truth™ is a think tank, curatorial group and creative studio dedicated to the critical analysis of notions of truth, fact, conspiracy, reality, and fake news. Our work is situated at the intersections of epistemology, politics, technology and aesthetics. We are based in Athens and London, with links to Berlin, New York, and New Delhi. The Ministry was formed in response to the ascendance of Donald Trump and the UK's exit from the European Union, two epochal events often considered the consequence of a new form of systematic misinformation. We investigate creative and strategic means of navigating the information apocalypse. The Ministry is an independent organisation with a network of collaborating institutions, universities and cultural foundations.
The Athens School of Fine Arts, established in 1837 at 42 Patission St, has ever since provided the infrastructure for the Arts in Greece.
Since 1992 most of the activities of the Athens School of Fine Arts moved to new premises at 256 Peiraios St, The factory (Εργοστάσιο).
The relocation in a new building that formerly housed the Sikiarides family textile factory, Hellenic Textiles SA, suggested a changed attitude but also new connections to the urban life. Most of the activities of the asfaBBQ will take place in this factory and more specifically in the Theater, the Cinema, the Garden and the classrooms of the School. In addition some of the events, will take place in the new project space of the School Circuits + Currents at 13 Notara & Tositsa St, Exarcheia and Entos/Ektos project at 44 Panepistimiou St.


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'The Under Presents: Tempest' Live VR Performance Is A Theater-Lover's Dream
CORRECTION [6/13/2020]: The Under Presents: Tempest is available exclusively on Oculus headsets, and not on PC VR as previously stated.
40 minutes of captivating theatrics brought to life by jaw-dropping visuals and interactive storytelling.
I'm standing at the center of a grand banquet table surrounded by a wide assortment of delicious foods and fine drink. Alongside me are a handful of other masked players, each of which partaking heavily in the bountiful feast provided to us by a mysterious omnipotent being. Just then, a towering figure appears in the sky above us and commands that each of us act as if we've lost our minds. The craziest performance would earn said player a mysterious prize.
I began to frantically wave my arms about the air as if possessed by the underworld's clumsiest ghost. Hopping onto the table I began tossing food throughout the dining area and even at the god himself. Pleased with my unstable behavior, my generous overlord dubbed the most unstable of the group, at which point I was awarded by hard-earned treasure.
The scene detailed above describes just several minutes of my time with The Under Presents: Tempest, a 40-minute interactive theater experience currently being performed live in VR via Oculus Rift and Oculus Quest headsets. Hosted on Tender Claws' immersive theater platform, The Under Presents, the interactive VR experience takes audiences through a one-of-a-kind live performance of William Shakespeare's classic play, The Tempest, completely reimagined for the VR format. Unlike conventional stage plays, The Under Presents: Tempest relies heavily upon audience participation; the players serve as everyone from the actors to the stage hands, resulting in an immersive theatrical experience unlike anything you'll find running on Broadway.
During a press event this past week, I had the opportunity to take the virtual stage alongside a handful of other theater-goers tuning in remotely from the comfort of their homes. Lead by a professional actor also turning in via VR, our group was transported from whimsical location to whimsical location as we acted our way through a uniquely-crafted rendition of the classic play.
At certain points it almost felt like performing a grade school play with some shockingly adult content matter. Taking the shape of several different characters throughout the course of the show, our host/director guided us through each seen, using a series of god-like powers to cycle through locations, trigger certain atmospheric effects, swap costumes, and perform a variety of other neat tricks to help better immerse users in the experience. This professional performer also serves as an acting coach, assigning characters and providing the motivations behind each action. Seeing as this is The Under Presents, however, players are unable to speak to one another' therefore quite a bit of body acting is involved.
During one portion of the show I auditioned for and was cast as Miranda, daughter of sorcerer Prospero, and was tasked with acting out the role of a beautiful maiden wandering throughout a serene forest. Naturally, I nailed my performance, acting out a series of seductive movements designed to attract the attention of the ultra-hunky Ferdinand, who was being played by another member of our group. While our director, Deirdre—shout out to Deirdre, she was absolutely amazing—guided us through the scene, the other two members of the group took on the role of the wind and performed a series of hypnotic gestures as we flirted awkwardly in VR.
Throughout the 40-minute performance, each of us played a major role in the story, offering just enough time in the spotlight to feel like a star. While I struggled to understand the appeal of The Under Presents upon its initial release back in 2019, this live theatrical performance shows just how powerful a tool VR can be in regards to storytelling. By combining the immersive capabilities of VR technology with conventional stage acting, Tender Claws is able to offer an interactive narrative experience impossible to recreate in a real-world venue.
Individual ticket prices feel a bit high at $15 per showing, but its important to remember that this is so much more than a passive viewing experience. Each session features a performance by a professionally-trained stage actor as they guide you through a fully interactive social/narrative experience. We're looking at the birth of a brand new genre of VR storytelling; one that makes you the star.
Whether you're a long-time theater geek or simply a fan of excellent storytelling, The Under Presents: Tempest is 100% worth the price of admission.
Showings run now through the end of September. Individual shows will take place from 4pm – 8pm PT on weekdays and 11am – 3pm PT on weekends. While The Under Presents itself is free (30 minutes of single-player gameplay included), players interested in catching a showing of Tempest will need to purchase a ticket for $14.99 via The Under Presents on Oculus Rift/Rift S, Oculus Quest, and SteamVR. Tickets also grant access to an exclusive multiplayer space.
Image Credit: Tender Claws
Multiplayer VR Oculus Oculus Quest Oculus Rift Oculus Rift S Social VR Tender Claws The Under Presents The Under Presents: Tempest The Under Presents: Tempest Review VR Live Show VR Theater
'Return To Castle Wolfenstein' VR Port Heading To Oculus Quest
Red Bull Flugtag VR Experience Gives You Wings

redpajama_set_name: RedPajamaCommonCrawl


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OILERS NOTES: Tough times for Coyotes and Golden Knights
Robert Tychkowski
Arizona Coyotes goaltender Carter Hutton (40) allows a goal against the St. Louis Blues during the second period at Gila River Arena, Oct. 18, 2021. Photo by Joe Camporeale /USA TODAY Sports
The list of things that aren't going according to plan for the soon-to-be-house-hunting Arizona Coyotes is long enough that goaltending isn't at the top of it, which is saying something when the starter gives up 14 goals on his first 47 shots.
It's early enough that the Coyotes (0-2-1 in their first three games) don't seem too concerned about it, but turning the goaltending reins over to Carter Hutton hasn't started out the way they'd hoped.
After losing Darcy Kuemper, Antti Raanta and Adin Hill in the offseason, the Coyotes brought in the 35-year-old former Buffalo Sabre to provide saves on the ice and mentorship in the room.
But Hutton gave up all eight goals in an 8-2 loss to Columbus in his Coyotes debut and six more in 36 minutes of a 7-4 loss to St. Louis. Backup Karel Vejmelka started their other game, a 2-1 shootout loss to the Sabres.
It's impossibly early to make any sort of determination — Hutton's 8.75 GAA and .702 save percentage over two games could change dramatically with one solid start — but the numbers don't look good in the here and now.
"If we give him proper support, we will be able to assess his work better," said Arizona coach André Tourigny. "I think it's unfair to assess the work of Carter Hutton and make that separate from our team.
"Two games he's been in, we had breakdowns for certain periods of time and that is where we got hurt. So we need to address that, and when we give him the proper support, I'm sure 'Huts' will be there, big and tall for us."
Tourigny said he started Vejmelka Thursday night against the Oilers for no other reason than that's the schedule he drew before the season started. It's not because he lacks faith in the other guy.
"Right now it's a topic, people ask that question, but I don't think it's on Carter Hutton," he said. "It's on everybody, coaches included, to be more consistent and have a solid 60 minutes."
Coyotes defenseman Jakob Chychrun adds that there is usually a lot that goes wrong before a goalie gives up a goal and in the case of the Coyotes, it has.
"We left him out to dry (against St. Louis). We all felt horrible because of it," he said. "You just hate to do that to a teammate. That can be a pretty lonely position when you leave a guy out to dry like that. We let him know we weren't good enough for him. It's as simple as that."
KNIGHT TIME
The Oilers are right back at it Friday when they hit the Vegas strip for what they hope is the first leg in a battle for first place in the division, and if there was ever a good time to get the Golden Knights, this is it.
With three of their best forwards — Max Pacioretty (broken foot), Mark Stone (undisclosed) and Alex Tuch (shoulder) — out of the lineup, the Knights might be ripe for the picking.
After a season-opening win over expansion Seattle, the Knights gave up 85 shots in their last two games while being outscored 9-3 by St. Louis and Los Angeles.
"It's not been good enough," said forward William Karlsson. "We need to be tighter, especially in the slot. We gave up way too many chances there."
It's going to be a challenge. Tuch is out three more months with shoulder surgery, Pacioretty is week to week and Stone is out indefinitely. But head coach Pete DeBoer believes they have the mettle in that room to fight back.
"My history with this group, when we've played a little bit scared of the situation or the opposition, we've responded," DeBoer said.
General manager Kelly McCrimmon is planning any panic moves to patch the temporary holes, either. It will be up to the remaining Knights to get this thing back on track.
"We sure won't be having any 'woe is me,' type of attitude," McCrimmon said. "Teams win short-handed. It heightens the importance of trusting your systems and provides opportunities for players that are going to get larger roles than what they would have if we were fully healthy.
"Sometimes, that's where hockey players are found."
LET'S PLAY TWO
During last year's North Division pandemic schedule players remarked how they really liked the idea of playing two road games in the same city and wanted to see the league adopt that as part of the schedule this year.
Naturally, the league ignored them, but playing back-to-back in either Glendale or Vegas instead of Glendale on Thursday and Vegas on Friday would have been nice.
"I enjoyed doing that last year," said Oilers forward Ryan Nugent-Hopkins. "I don't know if it's something they're seriously thinking about in the future, but last year I thought it was really good.
"You get settled into a hotel and you had a team lounge to hang out in and you're not travelling quite as much. I enjoyed it."
rtychkowski@postmedia.com
twitter.com/rob_tychkowski
Sign up to receive the Oil Spills newsletter, delivered straight to your inbox each weekday morning. Essential reading for Oilers fans who eat, sleep, breathe the orange and blue. To make sure our newsletter always reaches your inbox, please add oilspills@postmedia.com to your contact list or address book to keep us out of your spam folder.


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Home Gear / Parts Valentino Rossi 2020 MotoGP Yamaha Livery First Look: 18 Photos
Valentino Rossi 2020 MotoGP Yamaha Livery First Look: 18 Photos
Nine-time motorcycle Grand Prix world champion Valentino Rossi has revealed the livery for his 2020 Monster Energy Yamaha MotoGP Team YZR-M1 factory racer. Rossi is the most successful Yamaha rider in premier class history, with four MotoGP titles for Yamaha. 2020 marks Rossi's 15th season riding a Yamaha. Rossi has also ridden for Honda, where he won three MotoGP titles, and Ducati.
Although it has already been announced that 2020 is Rossi's swan song on the Monster Energy Yamaha MotoGP Team—he will be replaced by Fabio Quartararo—the nearly 41-year-old veteran Italian racer is determined to remain competitive. Rossi debuted in GP racing in the 125GP class in 1996.
"For some reason, the winter months always seem a lot longer than the summer months during the MotoGP season," Rossi observed. "Those seem to just fly by! Anyway, I'm happy to be on track and to start riding again. I've been able to relax during the stop, and also do some fun riding events, but now it's time to get serious about 2020. I'm very curious to see what steps we can make during the Sepang Test. For sure, Yamaha have prepared some new things for us to try and, anyway, the first laps of the year on a MotoGP bike after the winter break are always a unique sensation. It's always something I really enjoy. For sure, we have a lot of work ahead of us, but the team and I are ready for it."
After difficulties over the last two years for the Monster Energy Yamaha MotoGP Team, Managing Director of Yamaha Motor Racing Lin Jarvis is promising more for 2020: "If you scratch beneath the surface, you will find many changes that Yamaha and the team have made to improve the bike, the performance of our riders, and the total working efficiency of our team. We are excited to start this new season, and we believe we can achieve great things this year."
Team Director of Monster Energy Yamaha MotoGP Massimo Meregalli is also optimistic for the 2020 MotoGP season: "Yamaha's Japanese engineers have dedicated a lot of time and effort to developing the M1, and now our riders and engineers will put the new items to the test for the first time."
Valentino Rossi's teammate on the 2020 Monster Energy Yamaha MotoGP Team is Maverick Viñales, the 2013 Moto3 World Champion. Supporting the pair as the team test rider is three-time MotoGP World Champion Jorge Lorenzo, who retired at the end of last year's season. Lorenzo won his three MotoGP titles aboard a Yamaha.
Valentino Rossi 2020 MotoGP Gallery
Monster Energy Yamaha MotoGP Team
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Toys and Sporting Goods Products
WIMBIES
WIMBIES Trademark Information
Trademark by J.S. ENGLISH CO.
stuffed toy animals
This is a brand page for the WIMBIES trademark by J.S. ENGLISH CO. in *********, **********, ***** ****.
Write a review about a product or service associated with this WIMBIES trademark. Or, contact the owner J.S. ENGLISH CO. of the WIMBIES trademark by filing a request to communicate with the Legal Correspondent for licensing, use, and/or questions related to the WIMBIES trademark.
On Wednesday, February 22, 1995, a U.S. federal trademark registration was filed for WIMBIES. The USPTO has given the WIMBIES trademark serial number of 74637139. The current federal status of this trademark filing is CONTINUED USE NOT FILED WITHIN GRACE PERIOD, UN-REVIVABLE. The correspondent listed for WIMBIES is George L. Pinchak of ********,********, ***** **** . The WIMBIES trademark is filed in the category of Toys and Sporting Goods Products . The description provided to the USPTO for WIMBIES is stuffed toy animals.
Games and playthings; gymnastic and sporting articles not included in other classes; decorations for Christmas trees.
Word mark: WIMBIES
CONTINUED USE NOT FILED WITHIN GRACE PERIOD, UN-REVIVABLE
Goods and Services: stuffed toy animals
J.S. ENGLISH CO.
Mark Drawing Code: Typeset (Words/letter/Number)
George L. Pinchak
Trademarkia-Network law firms can help you incorporate a business around your WIMBIES trademark in less than 5 minutes. Trademarkia makes the process easy and convenient, so start now!
George L. Pinchak is a correspondent of WIMBIES trademark.
Please Rate and Review for WIMBIES
WIMBIES is providing stuffed toy animals.


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All issues Volume 41 / No 4 (July–August 2010) Vet. Res., 41 4 (2010) 47 Full HTML
Volume 41, Number 4, July–August 2010
https://doi.org/10.1051/vetres/2010019
Vet. Res. (2010) 41:47
Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals
Bettina Wagner1*, Alexandra Burton1 and Dorothy Ainsworth2
1 Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
2 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
* Corresponding author: bw73@cornell.edu
Accepted: 6 April 2010
Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-γ), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory TR1 cell phenotypes respectively, by flow cytometry. In agreement with previous reports, all three cytokines were quantitatively reduced in foals compared to adults. However, the balance between Th1 and Th2 cytokines (IFN-γ/IL-4 ratio) showed a clear Th1-biased response in foals by 6 and 12 weeks of life, while similar IFN-γ/IL-10 ratios were found in foals and adult horses. By day 5 after birth, intracellular IFN-γ production by foal CD4+ and CD8+ T cells resembled that in adults. Overall, IL-4 production was low in foals. IL-4+ cells peaked at day 5 of age when IL-4 was mainly produced by IgE+ cells. Relative percentages of IL-4+ Th2 cells were significantly lower in foals at all time points. The data suggested that equine neonates and young foals have an impaired Th2 response, that the immune response of foals is Th1 biased, that IFN-γ production by Th and cytotoxic T cells is qualitatively similar to adult horses, and regulatory IL-10 production by T cells is developmentally mature in foals during the first three months of life.
Key words: cellular immune response / cytokine / neonatal immunity / T cell differentiation / horse
© The authors, published by INRA/EDP Sciences, 2010
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted use, distribution, and reproduction in any noncommercial medium, provided the original work is properly cited.
After activation T helper (Th) cells develop into different subsets called Th1, Th2, Th17 and regulatory T cells (Treg). These T cell subsets express different cytokines and mediate distinct effector functions during immune responses. Th1 cells are characterized by interferon-gamma (IFN-γ) production and are required in defending against infection with many intracellular pathogens [36]. Th2 cells produce interleukin-4 (IL-4), IL-5, IL-13 and IL-31. They promote the development of antibody mediated immune responses against extracellular bacteria and parasites [27]. Tregs have major functions in maintaining peripheral tolerance, immune regulation, and in limiting inflammation during autoimmune diseases [39]. Treg cells express transforming growth factor beta (TGF-β) and/or IL-10 [14]. The most recently discovered Th cell subset is Th17 characterized by the secretion of IL-17. Th17 cells promote inflammation, play a crucial role in the clearance of pathogens during the host immune defense, and also induce inflammatory processes in autoimmune diseases [17].
In horses, Th cell subset detection was hampered by the lack of reagents to key cytokines characterizing the different Th cell responses. During the past few years, reagents to detect the production of several equine cytokines became available including monoclonal antibodies (mAb) to equine IL-4 [42] and IL-10 [43]. In addition, various groups have previously shown that a mAb to bovine IFN-γ cross-reacts with equine IFN-γ [32, 41]. This antibody enabled the detection of intracellular IFN-γ production during infection with equine herpesvirus type 1 (EHV-1) and equine influenza virus [6, 30]. Besides the lack of reagents to cytokines, Th cell phenotyping in horses is still restricted by the availability of T cell markers. Although antibodies to CD3, CD4 and CD8 were developed and characterized by two workshops on equine leukocyte antigens [18, 21], reagents for T cell receptors, natural killer cells or CD25 to better identify Treg cells are still missing. Thus far, the only Treg cell subsets that were characterized in horses were (1) a population of CD4+ T cells that simultaneously produces IFN-γ and IL-10 [43], and (2) a population of CD4+ foxp3 expressing cells that was recently characterized in peripheral blood mononuclear cells (PBMC) and endometrial cup cells [8]. The CD4+/IFN-γ+/IL-10+ Treg cell subset was named TR1 cells according to a similar cell population identified in humans and mice [14].
Many investigations have focused on differences in cytokine mRNA expression between equine PBMC isolated from neonates, foals and adult horses. Several parameters of the humoral and cellular immune response of foals were found to be decreased compared to that of adult horses [5, 7, 23, 33] causing some authors to conclude that the foal immune system is "immunodeficient". It has also been suggested that the reduced ability of equine neonates to produce IFN-γ and other pro-inflammatory cytokines enhances their susceptibility to intracellular pathogens [5, 7]. For instance, infection with the intracellular bacterium Rhodococcus equi causes severe respiratory disease in foals but mild or no clinical signs in adult horses [15]. However, in response to antigen stimulation, foal immune cells increased the gene expression of certain cytokines. After infection with R. equi, monocyte-derived dendritic cells from foals increased IL-12 mRNA expression more than cells from adult horses [11]. Foal PBMC also showed a clear up-regulation of IFN-γ mRNA, a decreased IL-4 gene expression and greater IFN-γ/IL-4 transcript ratios in response to R. equi infection compared to adult horses [16].
Similarly, IFN-γ production in response to EHV-1 infection was almost undetectable in foals but increased with age [29]. However, the IFN-γ producing cells in foals and young horses were identified as mainly CD8+ cytotoxic T cells (CTL) [31] which are considered to be associated with protection from disease [3, 19]. In older horses, the EHV-1 specific T cell response shifted towards a CD8− phenotype resulting in a clear decrease in IFN-γ producing EHV-1 specific CTL in aged horses [31]. These phenotypic variations in the IFN-γ producing effectors T cells were believed to offer some explanation for age-dependent differences in the susceptibility to clinical disease induced by R. equi or EHV-1 in foals and adult horses.
In addition, mRNA expression studies described a generally decreased ability of neonatal PBMC to express IFN-γ, TGF-β and IL-1α transcripts in comparison to cells from adult horses [5]. Other cytokines such as IL-8, IL-12 and IL-23 were found to be increased or similarly expressed in PBMC from neonates compared to older foals [20]. Stimulation of PBMC from foals with lipopolysaccharide (LPS) and IFN-γ also resulted in increased IL-10 mRNA expression compared to PBMC from adult horses [35].
These reports indicate that cytokine production and T cell development in foals likely vary depending on the cell type and the antigen-specific stimulus. Because the cytokine network is complex and interactions between immune cells can be manifold, a better understanding of the cytokine production by different immune cells of the horse is required. Here, we compared cytokine responses in foals and adult horses to obtain a broader understanding on the ontogeny of the Th cell response in healthy foals. We analyzed for the first time IFN-γ, IL-4 and IL-10 on a protein and cellular level using intracellular staining and flow cytometric analysis. We also investigated the development of Th1 cells (CD4+/IFN-γ+), Th2 cells (CD4+/IL-4+), the TR1 subpopulation of Treg cells (CD4+/IFN-γ+/IL-10+) and CTL (CD8+/IFN-γ+) during the first three months of life. Characterizing differences and similarities of adaptive immunity in foals and adult horses is important in assessing appropriate immune responses in healthy foals, in identifying inappropriate immune regulation in diseased foals, and in developing improved or new vaccination strategies for very young horses.
2.1. Animals and blood sampling
Heparinized blood sample were obtained via jungular venipuncture from 18 neonatal foals (day 1–2 after birth), 15 foals at day 5, and 15 foals each at 6 and 12 weeks of age, and 15 adult horses using the BD Vaccutainer system (Becton Dickinson, Franklin Lakes, NJ, USA). The horses consisted of Warmbloods, Thoroughbreds, and Thoroughbred crosses. The adult horse group was composed of 15 dams of the foals. The mares were between 8 to 22 years of age (median 14 years) and blood samples for this study were obtained at 12 weeks after birth. Foals were born and raised at the Cornell University Equine Park during the 2007 and 2008 foaling seasons. All animals were clinically healthy throughout the study. After birth, the foals suckled colostrum from their dams ad lib. On day 1, a blood IgG quantification was performed using the Snap Foal IgG test (IDEXX laboratories, Westbrook, ME, USA). All foals had serum IgG levels of > 800 mg/dL indicating sufficient passive transfer of maternal immunoglobulins.
All animal procedures were approved by the Cornell University Institutional Animal Care and Use Committee (Ithaca, NY, USA) and were in accordance with the guidelines established by the National Institutes of Health (Bethesda, MD, USA).
2.2. Cell isolation and fixation
PBMC were isolated from heparinized blood by density gradient centrifugation (Ficoll-PaqueTM Plus, GE Healthcare, Piscataway, NJ, USA). A total of 6 × 106 cells/well were cultured in 6-well plates in cell culture medium (DMEM containing 10% (v/v) FCS, 1% (v/v) non essential amino acids, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 50 μg/mL gentamycin) with Brefeldin A (10 μg/mL; Sigma, St. Louis, MO, USA) to block secretion of intracellular proteins. Aliquots of PBMC were either kept in medium or were stimulated with phorbol 12-myristate 13-acetate (PMA; 25 ng/mL) and ionomycin (1 μM; both Sigma) to provoke cytokine production. For a kinetic study on cytokine production, PBMC from four adult mares were stimulated with PMA and ionomycin for 2, 4, 6, 8, 18 and 24 h. All other stimulated samples and medium controls from foals and adult horses were incubated for 4 h in a CO2 incubator. After incubation, the cells were washed in PBS and fixed in 2% formaldehyde for 20 min at room temperature.
2.3. Intracellular cytokine staining and phenotyping of T cells
Intracellular staining was performed in saponin buffer (PBS, supplemented with 0.5% (w/v) BSA, 0.5% (w/v) saponin and 0.02% (w/v) NaN3). Monoclonal antibodies to equine IL-4 [42], equine IL-10 [43], and bovine IFN-γ (MorphoSys, AbD Serotec, Oxford, UK) were used for intracellular staining. The later antibody was previously found to cross-react with equine IFN-γ [32, 41]. A murine IgG1 isotype control was included in the procedure using an aliquot of the PBMC. After staining of around 1 × 106 fixed PBMC for 20 min at room temperature, cells were washed twice with saponin buffer and once with PBS/BSA (PBS, supplemented with 0.5% (w/v) BSA and 0.02% (w/v) NaN3). For cell surface staining of T cells, monoclonal antibodies to equine CD4 (HB61A, VMRD, Pullman, WA, USA), equine CD8 (CVS8, kindly provided by Dr Paul Lunn) and equine CD31 were used. Staining of IgE+ cells was performed with monoclonal anti-IgE 176 [40]. Cell surface staining was performed in PBS/BSA for 10 min at room temperature. Afterwards, the cells were washed once with PBS/BSA and measured by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA). A total of 30 000 events were measured per sample. All antibodies were conjugated to Alexa fluorochromes A647 or A488 (Molecular Probes, Invitrogen, Eugene, OR, USA) according to the protocol provided by the supplier, except for the FITC conjugated anti-bovine IFN-γ antibody.
A Shapiro–Wilk normality test was performed on each data set and showed that several variables were not Gaussian distributed. Thus, non-parametric test were used for data analysis. The foal groups at various time points were composed of animals living in the same environment but did not represent sequential samples. Cytokine production in the foal and adult groups and the cytokine ratios between age groups were compared by a Kruskal–Wallis test followed by Dunn's pairwise tests. For example, the total percentages of cytokine producing cells were compared at the lowest age to 2nd lowest, 2nd lowest to 3rd lowest, etc. For the cytokine ratios and the relative percentages of cytokine producing CD4+ and CD8+ cells, we compared each of the foal groups to the adults. The analysis was performed 2-tailed and with 95% confidence intervals. We used 2-sided alpha = 0.05 for significance and did not make additional adjustments for multiplicity beyond those implicit within each Kruskal–Wallis test and its Dunn's test. The statistical calculations were performed using the GraphPad Prism program, version 5.01 (GraphPad Software, La Jolla, CA, USA).
3.1. Cytokine production and T cell marker expression in PBMC stimulated with PMA and ionomycin
PBMC from four adult horses were stimulated with PMA and ionomycin for 2 to 24 h to identify the optimal stimulation time for cytokine production. Intracellular cytokines were detectable at all time points of stimulation (Fig. 1B) but not in non-stimulated cells (≤ 0.4% at all time points). The total percentages of IFN-γ or IL-10 producing cells showed a significant increase between 4 and 6 h of stimulation (IFN-γ p = 0.03; IL-10 p = 0.002) and reached a plateau afterwards. The percentage of total IL-4 producing cells also showed a tendency to increase during the first 6 h without being significantly different from the previous time point. Between 8 and 18 h, IL-4 decreased slightly but significantly (p = 0.03). For IFN-γ and IL-10, we also analyzed individual populations producing only one or both cytokines during 24 h of stimulation (Fig. 1C). Similar to total IFN-γ and IL-10 producing cells, the single IFN-γ+ or IFN-γ+/IL-10+ cells increased significantly between 4 and 6 h of stimulation. Single IL-10+ cells increased significantly between 2 and 4 h and then showed a tendency to increase without significant differences from one to the next time point and with increasing variations between horses as indicated by the high standard deviations at 18 and 24 h.
Flow cytometric analysis of IFN-γ, IL-4 and IL-10 producing PBMC after stimulation with PMA and ionomycin. PBMC were incubated in the presence of the secretion blocker Brefeldin A. Then, they were fixed and intracellular cytokine staining was performed. (A) Non-stimulated PBMC from a 6 week old foal after 4 h of incubation in medium with Brefeldin A. The left plot shows the gating (R1) on peripheral blood lymphocytes that was used for analysis of the data. The remaining three plots show a two-color staining of non-stimulated lymphocytes using anti-CD4 and different anti-cytokine antibodies. Typically, cytokines were not detected in equine peripheral blood lymphocytes in the absence of stimulation. Isotype controls for cytokine staining generally resulted in less than 0.05% of detectable cells. (B and C): PBMC from 4 adult horses were stimulated with PMA and ionomycin for up to 24 h and cytokine expression was measured at various time points. (B) Total percentages of IFN-γ, IL-10 and IL-4 producing cells in the lymphocyte population, (C) Percentages of IFN-γ+/IL-10+, IFN-γ+/IL-10− and IFN-γ−/IL-10+ cells during 24 h of stimulation. The data in B and C represent means and standard deviations. Differences in cytokine expression from one to the next time point were compared by Student's t-tests. ** p = 0.001 to < 0.01; * p = 0.01 to 0.05; (D) PMA and ionomycin stimulated cells from foals of different ages and adult horses. Double staining of IFN-γ and IL-4 or (E) IFN-γ and IL-10. The dot plots show one representative image from a foal or horse of the respective age group.
In addition, CD3, CD4 and CD8 expression was compared in stimulated and non-stimulated PBMC (Tab. I). In non-stimulated cells, the percentages of CD3+, CD4+ and CD8+ lymphocytes did not differ during 24 h of incubation. Previous reports have shown that PMA and ionomycin stimulation reduced the expression of CD4. Here, we observed that CD4 and CD8 expression decreased in PMA stimulated lymphocytes compared to non-stimulated cells, while CD3 expression was not effected by the treatment. Within the CD3+ cells, the percentages of cells expressing CD8 were up to two-fold higher in non-stimulated samples than after PMA stimulation. Significant differences in the stimulated samples were observed between 2 and 4 h (p = 0.0498), and 4 and 6 h (p = 0.0079). Longer stimulation did not alter the percentage of CD8+ cells any further. In contrast, the expression of CD4 decreased constantly in stimulated cells, with the exception of a slight increase between 4 and 6 h of stimulation (p = 0.0248). The ratios of CD4+ T cells in non-stimulated versus stimulated cells ranged from 1.37 at 2 h, 2.41 at 4 h, up to 9.11 at 24 h.
Table I.
Flow cytometric analysis of T cell marker expression in equine PBMC (n = 4) after stimulation with PMA and ionomycin (PMA) or in non-stimulated cells (med).
Because one major goal of this study was to investigate cytokine production in Th and TR1 cells, CD4 detection was crucial for the analysis by flow cytometry. All following experiments used PMA and ionomycin stimulation for 4 h to obtain sufficient cytokine production, while CD4 expression was still in an acceptable range. Although cytokine production and CD4 and CD8 expression were slightly higher in adult horses after 6 h compared to 4 h, we did not perform stimulations for 6 h because the cell recovery rate clearly decreased during the first 8 h of stimulation with PMA (data not shown) and cells of neonates were often very sensitive to the treatment resulting in even higher cell losses.
3.2. Cytokine production in foals at different ages
To analyze the production of IL-4, IFN-γ and IL-10 cytokines in foals during the first three months of life, PBMC were stimulated in vitro and intracellular cytokine production in lymphocytes was analyzed by flow cytometric analysis. Non-stimulated PBMC were run as experimental controls and did not contain detectable amounts of IL-4, IFN-γ and IL-10 (Fig. 1A) with the only exception being a small but distinct population of IL-4 producing cells evident in non-stimulated samples from 5-day old foals. PMA treatment of PBMC obtained from newborn and day 5 old foals resulted in low numbers of cytokine producing cells (Fig. 1D and E). Then, the total percentages of cells producing IFN-γ (Fig. 2A) and IL-10 (Fig. 2B) increased until 6 weeks of age and stayed at a similar level by 12 weeks of age. In contrast, the IL-4 production peaked in foals at day 5, decreased slightly but not significantly by 6 weeks and did not differ between 6 and 12 weeks of age (Fig. 2C). For all three cytokines analyzed in the foals, adult levels were not reached by 12 weeks of age.
Total percentages of (A) IFN-γ, (B) IL-10 and (C) IL-4 producing lymphocytes in neonates (n = 18), foals until 12 weeks of age (n = 15) and in adult horses (n = 15). PBMC were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and were analyzed by flow cytometry. The percentages include all cells producing the respective cytokine including double positive cells. The horizontal lines within the data sets represent medians. *** p < 0.001; ** p = 0.001 to < 0.01; * p = 0.01 to 0.05; ns = not significant.
3.3. Ratios of IFN-γ/IL-4 and IFN-γ/IL-10 in foals and adult horses
Cytokines are mediators of the cellular immune response and the balance of Th1, Th2 and Treg cells crucially influences the initiation and outcome of the immune response. One could hypothesize that comparable ratios of respective cytokines might result in similar immune responses in foals and adults, even if total numbers of cytokine producing cells differ with age. To analyze the relative concentrations of cytokines in foals and adult horses, we calculated the ratios of IFN-γ/IL-4 (Fig. 3A) and IFN-γ/IL-10 (Fig. 3B) using the total cytokine production in equine lymphocytes stimulated with PMA and ionomycin. The IFN-γ/IL-4 ratios in neonates at day 1–2 and day 5 after birth were similar to adult horses. At 6 and 12 weeks of age the IFN-γ/IL-4 ratios in foals were significantly increased compared to adult horses. In contrast, the IFN-γ/IL-10 ratios were not significantly different in neonates or foals compared to adult horses (p = 0.696).
Ratios of (A) IFN-γ+/IL-4+ and (B) IFN-γ+/IL-10+ lymphocytes in foals up to 3 months of age and adult horses. PBMC were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and measured by flow cytometry. The total percentages of IFN-γ, IL-4 and IL-10 producing lymphocytes were determined and ratios were calculated for the respective cytokines. The horizontal lines within the data points show the medians. *** p < 0.001; * p = 0.01 to 0.05.
3.4. Phenotyping of IFN-γ+ T cells
To investigate whether phenotypic differences of IFN-γ producing T cells could be identified between foals and adult horses, cells were double stained for IFN-γ and with CD4 or CD8 to identify Th cells and CTL respectively (Fig. 4). The percentages of IFN-γ+ cells within the two T cell populations were determined. Compared to adults, 1–2 days old foals had significantly fewer CD4+/IFN-γ+ cells (Fig. 4B) and a slightly increased but not significantly higher percentage of CD8+/IFN-γ+ cells (Fig. 4C). Five days old foals had a trend towards greater percentages of CD4+/IFN-γ+ cells and fewer CD8+/IFN-γ+ cells compared to adult horses, but these differences were not significant. The percentages of CD4+ or CD8+/IFN-γ+ cells of older foals were similar to adult horses.
Flow cytometric analysis of CD4+ and CD8+ IFN-γ producing lymphocytes. PBMC from foals of different age groups and from adult horses were stimulated with PMA and ionomycin. The cells were stained for intracellular IFN-γ and cell surface CD4 or CD8 expression. (A) Flow cytometric analysis of one representative horse per age group. (B) Relative percentages of CD4+/IFN-γ+ and (C) CD8+/IFN-γ+ T cells in foals and adult horses within the IFN-γ producing cells (= 100%). (D) Percentages of IFN-γ+/IL-10+ lymphocytes in foals and adult horses. (E) A tri-color staining was performed on stimulated PBMC to confirm that the majority of the IFN-γ+/IL-10+ cells are CD4+ T cells. Gates were set on lymphocytes (see Fig. 1A) and on IFN-γ+ cells (left panel). The IFN-γ+ lymphocytes were then analyzed for IL-10 and CD4 staining (right panel). The horizontal lines within the data sets B, C and D represent medians. *** p < 0.001; ** p = 0.001 to < 0.01; ns = not significant.
A third T cell population that simultaneously produces IFN-γ and IL-10 are TR1 cells. These regulatory T cells also express CD4. IFN-γ+/IL-10+ cells were clearly detectable in foals at 6 and 12 weeks of age (Fig. 4D) and represented the majority of the IL-10+ cells in foals and adult horses (Fig. 1C and E). Previous studies and data obtained here showed that 84.3 ± 3.3% of the IFN-γ+/IL-10+ lymphocytes are CD4+ TR1 cells (Fig. 4E). Within the total CD4+/IFN-γ+ cell population of adult horses, TR1 cells accounted for approximately 9%, while 91% of the CD4+/IFN-γ+ lymphocytes produced IFN-γ only, and thus represented Th1 cells. In foals, the relative amounts of IFN-γ+/IL-10+ cells within the total IFN-γ+ cells was 2.4% at 5 days, 6.9% at 6 weeks and 6.7% at 12 weeks of age. However, a significant lower amount of IFN-γ+/IL-10+ cells was only found for foals at day 5 of age compared to adult horses (p < 0.001). The relative amounts of IFN-γ+/IL-10+ cells from older foals did not differ from those of adult horses.
3.5. Phenotyping of IL-4+ cells
IL-4 producing cells in non-stimulated PBMC from foals directly after birth were previously shown to be CD4− cells. The IL-4+ cells had IgE bound to their surface and were characterized as basophils. To determine whether IL-4 production could be provoked in T cells from neonates and foals, PBMC were stimulated with PMA and ionomycin and double stained for intracellular IL-4 and cell surface CD4 or IgE.
PMA stimulation of PBMC from adult horses consistently induced IL-4 production in CD4+ T cells (Fig. 5) resulting in 87% of CD4+/IL-4+ cells and 5% of IgE+/IL-4+ cells. Although the total percentages of IL-4+ cells in foals were significantly lower than in adult horses at all time points (Fig. 2C), the percentage of CD4+/IL-4+ cells consistently increased from 8% at birth to 62% at 12 weeks of age (Fig. 5B). In contrast, the percentage of IgE+/IL-4+ cells progressively decreased from 95% in newborn foals to 17% at 12 weeks of age. In summary, PMA induced IL-4 production by Th cells was almost undetectable in newborn foals, when IL-4 was mainly produced by IgE+ cells. Then, IL-4 production by Th cells increased slowly with age. At 12 weeks of life, the relative percentage of CD4+ IL-4 producing cells had still a tendency of being reduced compared to adult horses. However, this difference was not significant anymore.
IL-4 producing CD4+ and IgE+ cells from foals and adult horses were measured by flow cytometry. PBMC were stimulated with PMA and ionocmycin. The cells were stained for intracellular IL-4 and cell surface CD4 or IgE. (A) A representative image from a horse of each age group is shown. (B) The total number of IL-4+ cells was normalized to 100% and the relative numbers of IL-4 producing CD4+ cells (%) were calculated for each horse. Medians are shown as horizontal lines within the data points of each group. *** p < 0.001; ns = not significant.
Cytokines are potent regulators of innate and adaptive immune responses. Their fine-tuning is crucial for the successful development of immunity and protection from disease. However, the interactions of cytokines with cells are manifold and result in a complex network of activating and suppressive mechanisms [1, 25, 27, 37, 38].
Cytokines produced by different Th cell subsets play major roles in orchestrating the adaptive immune response during infectious disease. The inability of foals to respond to infection or vaccination comparable to that of adult horses has often been interpreted to reflect "immunodeficiency" and was believed to be a reason for the increased susceptibility of foals to intracellular pathogens such as R. equi [5, 7, 20]. Other authors have suggested that foal immune system exhibits a Th2-bias [5, 23, 26]. This conclusion might have been drawn from a paradigm in neonatal mice indicating a Th2 dominated immune response, although strong Th1 responses have been induced depending on the experimental conditions [4, 12, 13]. In contrast, data on human neonatal T cells did not support that T cells are Th2 polarized and rather point towards a Th1 or Th0 type response in human neonates [2, 9, 22].
In this study, we analyzed the protein expression of three key cytokines, representing Th1 (IFN-γ), Th2 (IL-4) and TR1 (IL-10 and IFN-γ) responses, to investigate the development of Th cell responses in foals during the first three months of life. Previous studies on the induction of adaptive immune responses in foals and adult horses have focused on the expression of mRNA [5, 11, 16, 20, 35] but failed to provide detailed information about the cellular source of cytokine production. For example, IFN-γ mRNA up-regulation in PBMC could originate from several cell types such as Th cells, CTL or NK cells and would not necessarily represent a Th1 response. Similarly, other authors have analyzed the production of a single cytokine (IFN-γ), at the protein level with or without regard to the cell type [7, 29, 31]. This work has shown that IFN-γ protein and mRNA expression after PMA stimulation of PBMC from foals attained adult levels at around one year of age [7]. We also observed reduced IFN-γ production in neonates and older foals compared to adult horses after PMA stimulation. Similarly, the IL-10 and IL-4 production was clearly reduced in neonatal and foal PBMC when compared to adult cells. This showed that the PMA induced production of all three cytokines, IFN-γ, IL-4 and IL-10, is quantitatively lower in foals than in adult horses.
PMA is a structural analog to diacylglycerol and activates protein kinase C (PKC). PMA can activate different cell populations via this pathway, including T cells, B cells, basophils, mast cells and various other cell types. In T cells, the same intracellular signaling pathway is used following antigen-specific T cell receptor (TCR) activation resulting in T cell differentiation and cytokine production. Similarly, PMA can activate T cells independent of their TCR specificity by directly inducing PKC and cytokine production [45]. Thus, PMA stimulation of PBMC results in a polyclonal cytokine response of the naïve T cell pool, yielding insight into the general potential of these cells to produce cytokines. However, the quantitatively reduced cytokine response to PMA in foals compared to adult horses does not necessarily mean that antigen-specific T cell activation would also result in a non-protective immune response in foals. For example, by comparing responses to R. equi infection in healthy foals and adult horses, monocyte-derived dendritic cells from foals responded with increased IL-12 mRNA expression, the innate cytokine which triggers Th1 cell development [11]. Healthy foals also showed an up-regulation of IFN-γ mRNA, a decreased expression of IL-4 and increased IFN-γ/IL-4 transcript ratios in response to R. equi [16]. In another pathogen model, the IFN-γ production in response to EHV-1 was found to be low in foal PBMC but the EHV-1 induced IFN-γ producing cells were mainly protective CD8+ CTL [29, 31]. In contrast, aged horses which have a higher susceptibility to EHV-1 infection, showed a clear decrease in IFN-γ producing EHV-1 specific CTL [31]. Thus, the antigen-specific T cell response to a specific pathogen can still be sufficient and protective in the foals even if the response varies quantitatively from adult horses.
Twenty years ago, Mosmann and Coffman [27] published their famous studies on the induction of Th1 and Th2 pathways in the Leishmania major model in mice. Since then, it is well accepted that the balance of Th cell cytokines is crucial for the outcome of the adaptive immune response and the development of immunity and protection to infectious diseases. Today, this general concept still has important implications for vaccine development. Classic vaccine formulations were often found to be less effective in young foals. To develop protective vaccine strategies for foals early in life, it is important to understand differences and similarities of neonatal and adult immune responses in healthy horses. One way to analyze qualitative parameters of the adaptive immune response is to determine the balance of cytokines produced by Th cells. Despite the quantitative reduction of IFN-γ produced by foal CD4+ cells compared to adult cells, our data demonstrated that the balance of Th1/Th2 cytokines in foals was highly dependent on age and biased towards the Th1 phenotype. A reduced Th1/Th2 cytokine profile compared to adults was only observed on day 5 after birth and was mainly due to the peak in IL-4 production by IgE+ cells. In 6 and 12 weeks old foals, the T cell response showed a clear Th1 dominated cytokine profile. In addition, adult-like IFN-γ/IL-10 ratios and the IFN-γ production by Th cells or CTL were evident within 5 days of birth. Collectively these data demonstrated that T cells from equine neonates and foals are competent in mounting Th1, CTL and TR1 responses that are qualitatively similar to those observed in adult horses.
In addition, we found that equine neonates and foals had an impaired Th2 response that did not reach adult-like levels even by 3 months of age and that the IL-4 production in neonates and very young foals originated mainly from IgE+ basophils and not from T cells. These data challenge the dogma that equine neonates and foals mount Th2 biased immune responses [5, 23, 26]. Similarly, non-stimulated neonatal basophils isolated around day 5 after birth produced IL-4 and increased IL-4 secretion from these cells was observed after stimulation with anti-IgE [44]. Recent reports derived from humans or experimental mouse models provide increasing evidence that basophils are the most important innate inducers of Th2 cell differentiation during allergic conditions and responses to parasites by providing the early IL-4 signal that initiates the polarization of Th cells towards the Th2 phenotype [10, 24, 28, 34]. Similarly, basophil derived IL-4 could provide the innate trigger to initiate Th2 differentiation in equine neonatal T cells. However, although IL-4 from basophils was detectable during the first week of life, we observed a clear delay in the development of IL-4 producing Th cells in foals. Thus, an immediate or broad influence of the early IL-4 production by basophils on the development of Th2 immunity is rather unlikely.
Overall, the T cell response in neonatal foals to PMA reported here and by others using antigen specific stimuli [16, 29, 31] more closely resembled immune responses in human as compared to murine neonates. Human neonatal T cells were also found to develop weak IL-2 and IFN-γ responses and proliferated poorly in response to TCR mediated stimulation. However, if human neonatal T cells were stimulated by TCR-independent pathways, such as crosslinking of CD3 plus recombinant IL-6 or IL-12 or anti-CD28 antibody, adult-like T cell responses were observed [2].
In summary, our results provide a new point of view on the development of Th cell responses and the balances of Th1, Th2 and TR1 cytokines in healthy equine neonates and foals.
We would like to thank Ms. Carol Collyer and the crew of the Equine Park at Cornell University for their help with obtaining the blood samples for this project. We also thank Ms. Bronwen Childs, Ms. Julie Hillegas and Ms. Esther Kabithe for excellent technical assistance. Funding for this project was provided by the Dean's Fund for Clinical Excellence at Cornell University.
Wagner et al., unpublished data.
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Protection, systemic IFN$\gamma$, and antibody responses induced by an ISCOM-based vaccine against a recent equine influenza virus in its natural host
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Which Departing 'Shameless' Cast Member Will Return for Season 10
Shameless fans said "goodbye" to the glue that the Gallagher family often held together. As she tried to rise in Chicago's Southside, Fiona Gallagher, played by Emmy Rossum, decided it was time to break free.
Rossum closed the season, her departure being the main focus of the season finale of season 9. Now that she no longer had to be the family mother, Fiona failed, which meant interference with the law. Her lawyer and her brother Ian advised her to leave the area and eventually grow up. So she did.
Although Fiona may have flown to the south (because she said she had the winters in Chicago), will she be back? Or will other characters who were planning to leave be back?
This is what Rossum says about returning next year
Rossum spoke to Entertainment Weekly about the evolution she was going through during her tenure as shameless. "I was 23 when the show started and it was quite remarkable the trust it gave me," Rossum said. "It has been a long, beautiful journey and I am so close to my Gallagher family that it is pretty bittersweet to walk away, but it felt like it was time for the character to spread her wings and that there was less need for hair, I never want something to just feel like a job and so I leave while I still love it. "
At the age of 32, Rossum said she wanted to leave her character on the resurgence. "I've always wanted Fiona to go to a high level and I've campaigned from the beginning, Fiona is not just in my heart, but I think it's kind of a pillar of power in the family," she said.
Final episode tonight. Here is a picture taken eight years ago during our pilot. Today, 110 episodes later, I am filled with gratitude for my shameless family and for our faithful audience. I'll be watching with love and can't wait to see what comes next. ❤
A post shared by Emmy Rossum (@emmy) on Mar 10, 2019 at 9:32am PDT
So is the door still open for a possible guest star return? "I would never close my door for the family," she said. "As I said in what I've written and what I've said to them repeatedly, they just have to think that I'm in trouble." As she says, never say never, she is not sure how much more story about Fiona should be told.
But this character will return
Actor Cameron Monaghan, who plays Ian, also announced that he would not return to the series either. Monaghan stepped back from Shameless earlier this season after Ian was sentenced to jail. And while he no longer performed, he appeared in the final of season 9 to give Fiona advice on leaving the city.
Although he had already announced his departure, Monaghan agreed to resume his role as Ian for season 10. He told ET why he decided to return. "I took some time away and spoke to John Wells, the show runner, about possible things we could do with the character in the future," he said. "And we came both creatively and financially in a place where I felt comfortable coming back.Much of that had to do with what we were going to do with the story, so I'm very glad I can go back and do that . "
Sis. ❤ #Shameless
A post shared by Cameron Monaghan (@cameronmonaghan) on Mar 10, 2019 at 8:14pm PDT
Monaghan confirmed that his character will return for the entire season, but added that he has only been registered for one season. But "It does not mean I'm against it [extra seasons] It's just … it's just as creative as it is," he added. "It's just doing business well and taking care of what I want to do as an actor."
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This Toxic Herbicide Was Found In Breast Milk