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Analysis Snakes in the Grass: Who is Benefiting From Joey Gibson's Rallies?
Snakes in the Grass: Who is Benefiting From Joey Gibson's Rallies?
Seattle GDC
On Christmas Eve, 1985, David Lewis Rice murdered Annie, Charles, Colin, and Derek Goldmark at their home in Seattle because he mistakenly thought they were Jewish communists. He was an active member of a Christian Patriot group called the Duck Club, who promoted the anti-Semitic and anti-communist views he claimed as motivation for stabbing and beating a family to death on the eve of a major Christian holiday. The group maintained its innocence of any incitement to violence, but they never explained why he was involved with the group. What attracted a disturbed, violent individual like David Lewis Rice to a group that espoused anti-communist and anti-Semitic conspiracy theories under the guise of Christian values?
On May 26, 2017, Jeremy Christian killed two people and wounded a third who interceded when Christian was verbally assaulting two girls with Islamophobic and racist speech in Portland, Oregon. On April 29, he had appeared at a 'March For Free Speech' led by the Vancouver, Washington-based group called Patriot Prayer. After Christian made several Nazi salutes and shouted epithets, Patriot Prayer organizer Joey Gibson told Christian to leave, but he's never been able to explain why Christian was there. What attracted him to Patriot Prayer, a group that espouses anti-communist and Islamophobic rhetoric under the guise of Christian values?
Joey Gibson has attempted to sell a candy-coated form of Christian Patriotism to the mainstream, but his attempts at inclusivity and 'dialogue' are clearly duplicitous. Now that the constant violence at his rallies has alienated his more mainstream fans, Patriot Prayer is moving even further to the right. Patriot Prayer continues to attract white nationalists, anti-Semites, homophobic street preachers, and self-described fascists to their events, while overtly propagating Islamophobia at events like ACT for America's March Against Sharia Law. Joey Gibson has made little attempt to distance himself from his more extreme fans, and it's clear that he's in no hurry to tell them to leave now. Meanwhile, he continues to avoid the question: Why are they there?
Gibson (bottom left) posing with neo-nazi Identity Europa organizer Jacob Von Ott on Sunday 8/6/17.
The truth is that Patriot Prayer isn't coming to Seattle on Sunday 8/13 to promote Christian values. Joey Gibson does not want dialogue. He wants confrontation. He wants to abuse and assault anyone who doesn't believe his endless stream of lies. It's time to show him and his friends how wrong they are.
He's even adopted the same Feds-and-Reds conspiracy theory that has driven countless right-wing Sovereign Citizens and Christian Patriots to violence and murder. This week, he wants it to be a freedom rally again, and he's talking about Jesus for the first time since he entered the regional consciousness as an 'activist' for a cause he can never quite define. His core values are no longer his own, and his sad but predictable path of self-brainwashing will only lead to more dangerous and unpredictable behavior. Meanwhile, his friends aren't going anywhere. So we find ourselves asking, "Who is benefitting from Joey Gibson's rallies?"
Bruce Cunningham Jr.
Nice nazi hat Bruce!
Bruce is an area neo-nazi based out of Shoreline, WA and is a perfect example of the type of dangerous individual that Joey Gibson is more than happy to create space for at his events. Bruce enjoys sharing nazi and other white nationalist imagery and is very open about it as evidenced by a cursory glance at his social media. Notice the Confederate flag life rune and US flag Sonnerad in addition to the multiple pictures of nazi planes and gear that he wears.
His Facebook profile is almost entirely white nationalist pictures and fake news stories.
Despite the above-ground nature of Cunningham's neo-nazi ideology, Joey was happy to stand alongside him at the Patriot Prayer "Anti-Communism" event at Westlake Park on May Day. Bruce was thrilled to report in the comments of a video from that day that he was able to save a "Kekistan" flag. Given that the flag is a near-replica of the nazi flag, there is little surprise there.
Bragging about fighting on May Day to save a Kekistan flag.
His Youtube account includes an uploaded tribute to nazi Germany.
Cunningham Jr. on May Day after snatching back the Kekistan flag.
Considering how often Joey Gibson claims that his events are not white nationalist events, it is alarming that he continues to host events that this individual feels safe to attend. The fact that the amorphous ideology Gibson claims to be spreading to his events appeals so strongly to people like Cunningham is a threat to our community that cannot be ignored.
Bruce simply cannot wait for the event on A13!
Proud heathen, sure. But what kind of heathen?
Audina Wharton
Audina Wharton is another self-identifying neo-nazi from the area (originally from Selah, WA but may live in Tacoma now) who keeps showing up at Joey Gibson's events. A cursory look at her Facebook profile shows that not only is she friends with Bruce Cunningham Jr., but that she also gladly identifies as a "beautiful Aryan woman".
Her Facebook profile, much like Cunningham Jr.'s, includes nazi imagery. One recent development with Audina is the fact that she appears to be engaged on some level with The Asatru Community. Within the pagan world, there has been an ongoing struggle between the disgustingly racist Asatru Folk Assembly and The Asatru Community (TAC). With TAC declaring themselves unaffiliated with the hideous ideologies of the Folk Assembly earlier this year, one has to wonder how they feel about somebody who claims affiliation with them also sharing neo-nazi pictures and memes.
Rose City Antifa researchers report that "she has close family members active in white nationalist prison gangs, and her Facebook friends list is a who's who of well-known violent neo-Nazi skinheads (boneheads) from American Front, Hammerskins/PNW Wolfpack, Volksfront, and other groups like Cascade Legion, Wolves of Vinland, and more."
Dan "Duerst the Wuerst"
Dan Duerst is not affiliated with any particular group as far as we know, but we included him in this report because he has been seen attending and engaging with a variety of Proud Boys and other alt-right individuals at actions in the area recently. He runs a YouTube page and is usually filming at actions, doing his best to obtain footage of antifascists and other counter protestors to edit and upload later for sharing on 4chan and other alt-right online hangouts. He should be considered a doxing threat and avoided if possible.
Cascade Legion group shot including (L to R) Michael Kral giving a nazi salute, "Seff Kalki", Huges, and Thorfin.
The Cascade Legion
The Cascade Legion is a relatively new formation, which coalesced earlier this year in response to a notion of a perceived victimhood at the hands of "Antifa terror" and "disgusting displays of anarchy." Their membership varies from actual neo-nazis, such as Seattle's own Charles Krafft, to internet meme kids, to Odinists and fascist heathens. The stated goal of the group is to provide defense to the right wing and assisting the police in engaging in political repression of their opposition. Several of their members have RSVP'd to Joey Gibson's event on the 13th who we will cover next.
Neo-nazi Charles Krafft showing his affiliation with the Cascade Legion.
Jeffrey Hughes
Jeffrey Hughes is associated with the Cascade Legion, an Oathkeeper, a Mason, and a self-avowed fascist. Our friends at Rose City Antifa recently released an extensive report on him which can be found here. He frequently discusses his goal of uniting the police and militia organizations with the white nationalist movement, showing either his influence on the Cascade Legion or vice versa. Please take the time to read the entire Rose City report; it paints a crystal clear picture of what kind of person Hughes is and how he continues to benefit from the events Joey Gibson organizes.
Thorfin Odinson
Thorfin who lives in Longview, WA is a member of the Cascade Legion and the most obviously-Odinist member. Odinson also appears to be involved in the III%'er movement to some extent. A cursory look at his social media presence shows that he has been in attendance at several Joey Gibson events in Portland and Olympia and includes memes such as the one below which shows a heathen and crusader teaming up to fight Black Lives Matter, feminists, and a variety of leftists being controlled by a caricature of a Jewish person with a Sonnerad overhead.
How much shitty stuff can we fit on a meme? Let's find out.
Thorfin and his militia buddies.
Garry Bergstrom
Garry Bergstrom from Kennewick, WA is buddies with Thorfin Odinson and another member of Cascade Legion. He has made appearances at other Joey Gibson events such as his anti-communist one here in Seattle on May Day. He shows up at actions as "Red Skull", because nothing says bad-ass quite like wearing a shitty plastic Halloween mask.
Garry showing off a Viking battle axe with the Cascade Legion Flag behind him.
David Fry
David Fry of Lakewood, WA is the president of the American Freedom Motorcycle Club which is affiliated with the "American Freedom Warriors" formation which is the loose coalition of various violent individuals and groups that has coalesced around Joey Gibson and his traveling circus. Fry has traveled as far as Berkeley to chase down and assault protestors in the streets.
Fry choking and beating a protester on the ground in Berkeley.
However, just a few days ago David took to his Facebook to denounce Gibson's planned event in Portland due to his continued cooperation with fascists such as Identity Europa. While his actions in Berkeley speak for themselves, we genuinely hope and want to believe him when he says he has turned over a new leaf and is done with enabling these fascists to freely organize. That is, after all, our goal here; to prevent people from being duped into helping create a safe space and organizing opportunities for abject fascists.
Only time will tell if David is serious about this.
Aaron Laigaie
Aaron Laigaie is one of the co-founders of the "Mount Baker Proud Boys" out of Bellingham, WA and an Army veteran. The Proud Boys are a disturbingly misogynistic, Western Chauvinist group which chooses to hate based more along the lines of hyper-masculinity and an ahistorical notion of the West rather than the more common racial lines that hate groups draw and identify with. On June 10th, during the ACT for America anti-Muslim rally a group of Proud Boys repeated pursued and attacked the peaceful counter protest. In addition to this, Proud Boys just began posting their own propaganda for the first time here in Seattle.
Aaron shares a pic of some Proud Boys in Portland on 8/6 and promises more for 8/13.
Poster from the first wave of Proud Boy propaganda found in Seattle, August 8th.
The Proud Boys also are advocates for men refusing to masturbate. This ties in to a deep anxiety around the idea that "Western" countries are not having enough sex to keep up with other cultures (primarily Muslims). Don't believe us? Hear it from Aaron himself:
Anthony Edward Bell
Anthony Edward Bell is another one of the Mount Baker Proud Boys. His obsession with Christian crusaders is a common theme amongst the Proud Boys and the broader alt-right and alt-lite and speaks to the same racial and cultural anxiety that drives their desire to not masturbate to increase their virility. They have been recruiting fairly steadily up there and their growth presents a threat not only to their own community but every community along the I5 corridor. Bell has been spotted as far as Portland at other Joey Gibson events.
Bell picture with "Based Stickman", an individual with a long and dark history of violence.
Jake Farmer
Jake Farmer is another Proud Boy, this time out of Vancouver, WA.
Bentley posing with 'Seff Calki', a member of Cascade Legion, outside Milo's tour bus at UW on J20
Kory C. Bentley
While Kory Bentley is not known to be officially affiliated with any particular crew, it is clear that he falls somewhere on the spectrum between alt-right and alt-lite and associates on some level with local Proud Boys and members of the Cascade Legion. His profile showcases some of the same, tried crusader memes that other Proud Boys share. He tried (and failed) to attend the Milo talk at UW choosing to instead then go online to lie about what had happened that night, telling his network that one of his friends had been shot. Given that the victim of that night was one of our own, we'd like to kindly tell Kory to fuck off.
Bentley with some friends at Finn's on University Ave either before or after the Milo talk on J20.
https://www.greaterseattlegdc.com/
Seattle branch of the General Defense Committee (GDC) of the Industrial Workers of the World (IWW).
Portland, OR: Vigil Outside of ICE Detention Center July 17th
Olympia, WA: "We Are The Fire That Will Melt ICE"


Korean buckwheat noodles are much the same as Japanese soba, a thin noodle, which can be made entirely from buckwheat flour, or more commonly mixed with varying quantities of wheat flour. Despite the name buckwheat is wheat and gluten free. They can also be used in the 'chilled buckwheat noodle' dishes.


text:
Hilton Head town board approves Shelter Cove mall design
By BRIAN HEFFERNAN
bheffernan@islandpacket.com
Published Tuesday, February 12, 2013
Read Complete Article Here
Developers won final approval Tuesday to build a new outdoor mall to replace The Mall at Shelter Cove.
After examining landscaping drawings and brick samples from architects Wood + Partners Inc., the Town of Hilton Head Island's Design Review Board unanimously approved the plans with only minor stipulations.
Tuesday's endorsement allows developers -- Blanchard & Calhoun Commercial -- to proceed with the Shelter Cove Towne Centre, a mixture of shops and cafes that will be a part of the revamped mall.
Demolition began Jan. 10, and new construction could begin within the next 30 days.
Plans call for an outdoor mall with several single-story shops and cafes, set between a Kroger grocery store on one end and the existing Belk department store on the other. A walking area leading to views of Broad Creek will bisect the mall.
The project is expected to cost about $40 million and feature 290,000 square feet of retail space, according to Mark Senn, president of Blanchard & Calhoun Commercial.
Construction will be completed in two phases. The first covers the northeastern half of the mall where the Kroger will be located. It is expected to be completed in the fall, Senn said.
The second phase covers the southeastern half of the project and is expected to be finished in the spring or summer of 2014, he said.
The project also includes plans for 210 apartments with views of Broad Creek. Shelter Cove Community Park would be relocated to land between the shopping center and the creek. This final portion of the project must still win town approval. If it does, work would be completed in the fall of 2014.
That part of the plan is expected to cost about $20 million, Senn said.
The town has agreed to pay for half the estimated $4.5 million cost of the park, but will pay no more than $2.25 million if the project goes over budget.
On Tuesday, the board seemed to favor the design features, which included indigenous trees, benches, a bike service station, drinking fountains with a dog-level fountain and surfaces made up of concrete mixed with oyster shells.
The plan passed with several minor conditions:
Fences that hide shop loading docks will be made of wood or masonry.
Trash bins will match the color of adjacent buildings.
The future design of retail kiosks and two specific store fronts will be reviewed by the board.
Developers will consider landscape recommendations from the board once the project is completed completion.
Board members Todd Theodore and Tom Parker Jr. abstained from voting -- Theodore works for Wood + Partners, and Parker, an island architect, was a design consultant.
Newer PostShelter Cove Towne Centre Groundbreaking
Older PostPHOTO: Demolition begins at Shelter Cove Mall on Hilton Head

redpajama_set_name: RedPajamaCommonCrawl


Long time readers over here will remember the time, when I'd only been blogging for a few months, my daughter and her BFF sat down to warn me about the dangers of talking to people on the internet– specifically referring to what my kids called my "Imaginary Internet Friends." It was pretty funny, go read it if you haven't.
In other news, July is gone and we're well into August and, yes, I'm still on hiatus from social media. Mostly.
It took me a good week to get over feeling "deprived" of input, but the days since then have been calmer and more peaceful. I've been spending less time online, in general. The only thing I keep open and respond to in real time is messenger, and only because that's mostly how my kids communicate with me. I don't ignore them. Ever.
The strategy has been effective, although I can't say I've been absolute about it. It's not quite the extreme of "head in the sand." It's more like sitting on the sand and contemplating the ocean while chaos reigns inland, occasionally glancing at it over my shoulder.
I do sometimes check in to see what friends are posting on FB, since very few of them post angsty political things. Every once in a while I'll briefly skim to see what the outrage du jour is over on twitter. But I set a timer and am strict with myself about shutting it down once time is up.
It's been refreshing, this feeling of detachment and the lack of noise.
What have I been doing? A lot of reading. A wee bit of writing. Catching up on small non-urgent stuff around the house that I'd been neglecting, as well as a couple big things. Being more mindful about getting in my steps every day. Listening to the thunder and rain during what has been an unusually wet summer. Enjoying my granddaughter.
She's so expressive for her age.
In fact, she expressed all over her diaper just before this was taken, explaining the satisfied smile.
And really, I've been doing a lot of sittin' and thinkin' — which is death when characters in a novel do it, but is apparently quite necessary for the writer. This writer, anyway.
The upshot is, I'm starting to feel little sparks of creativity amidst moments of nascent optimism. So I've decided to continue along these lines for a while longer.
I'm seeing triple, but I've been drinking.
Do you feel like a trendsetter, having imaginary friends before your kids did?
She is beautiful!! Love hearing the sparks are showing some lovely lights!
Thanks, all. I think she's beautiful too, but I realize I'm not even slightly objective about it.
They all came for a nice long visit today, as they had errands to run in my area, and she was very talkative. Had all sorts of things to discuss with me. Her parents said it's the most talkative they've seen her. Such a treat. Of course, then she got tired and crabby and cried her little heart out, poor thing, until she fell asleep.
She spit up on me and now my shirt smells like baby and I don't even want to change. But I do think I might need a nap.
An axe murderer? Highly unlikely, Evelyn. But if you are, I think I know you well enough after all these years to be extremely confident that, well, the axe had it coming. You might even say it was axing for it. And if it had handled things better, it wouldn't have had to steel itself for its inevitable demise.
I'm trying to think up a cutting response, but it's Monday. My wits are not sharp enough.
I understand. It takes years (and lots of nonsense) to hone this brand of trenchant humour.


We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12).
DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells.
Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis.
In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.
Rheumatoid arthritis (RA) is a systemic disease consisting of chronic inflammation in multiple articular joints. Current treatments such as systemic anti-tumor necrosis factor (TNF)-α therapies are effective in > 50% of RA patients ; however, biologics must be given in relatively high dosages, and significant side effects have been reported . The success of the best-known altered peptide ligand, Copaxone (Teva Pharmaceuticals, Petach Tikva, Israel.) as a first-line therapy for multiple sclerosis (MS), suggests that peptide therapies based on naturally occurring proteins may provide an effective alternative to drug biologics for the treatment of arthritis. Although various strategies have been tested, we have developed a unique modification to a protein that is the predominant collagen type found in human cartilage. Our therapy is based on our previous discovery of an analog peptide of type II collagen (CII) that could profoundly suppress arthritis in the CIA model by inducing a unique inhibitory T cell .
Although the original observations were made by using DBA/1 mice, we have extended our findings to a humanized animal model specifically developed to mimic more closely the pathogenic features of RA. In this model, mice transgenic for one of the most common RA-susceptible major histocompatibility complex (MHC) molecules, DRB*0101 (DR1) [4, 5] become arthritic when immunized with CII/CFA. By using proliferation and cytokine assays, we determined that a peptide representing CII263-270 and containing amino acid substitutions at positions 263 (F→N) and 266 (E→D) (analog peptide A12) was profoundly suppressive, effectively attenuating arthritis in the humanized RA-mouse models, despite having very poor avidity for the MHC .
Although collagen peptides designed with carefully crafted substitutions such as A12 may provide an attractive alternative choice for treatment of RA, the low numbers of antigen-specific T cells in wild-type mice have made functional T-cell studies in autoimmune arthritis particularly challenging. Studies using TCR transgenic T cells are limited to the study of one TCR, and the low MHC binding avidity of peptide A12 makes untenable the strategy of loading empty class II molecules with exogenous peptide to be assembled as MHC multimers (tetramers). To overcome this problem, we generated DR1 multimers [7, 8], in which the analog peptide A12 was covalently linked, making it more likely that majority of the MHC molecule binding clefts are occupied by this peptide [9–11]. By using the tetramers as a tool to study analog-responsive T cells, we immunized DR1 tg mice with A12/CFA and isolated draining inguinal lymph node cells. Culture with the tetramer revealed that A12-specific T cells were readily detectable. Moreover, the A12-responsive T cells could be visualized to characterize the TCR-Vβ repertoire and cytokine secretion profiles. Adoptive transfer experiments demonstrated that tetramer+ cells were very effective in preventing and suppressing inflammatory arthritis.
Looking to the future, class II tetramers, such as DR1-A12 show promise for monitoring the development of analog-specific T cells in clinical settings whenever low-avidity analog peptides will be used to treat patients with RA.
Native CII was solubilized from fetal calf articular cartilage by limited pepsin-digestion and purified as described earlier . The purified collagen was dissolved in cold 20 mM acetic acid at 4 mg/ml and stored frozen at -70°C until used. The synthetic peptides were supplied by Biomolecules Midwest Inc. (Waterloo, IL, USA). A peptide containing the immunodominant determinant sequence of CII (GIAGFKGEQGPKGEB) is referred to as A2 or wild-type (WT) IEDBID 109115; a synthetic peptide representing the sequence (GIAGNKGDQGPKGEB) is designated A12.
Mice expressing the chimeric (human/mouse) DRB1*0101 construct were maintained in our onsite pathogen-free facility. The chimeric DRB1*0101 construct has been previously described, as has the production of Tg mice expressing these constructs .
The IL-4 (C57BL/6- IL4tm1cgn IL-4 and C57BL/6- IL10tm1cgn knockout) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and backcrossed onto the arthritis-susceptible DR1 C57BL/10 background for 12 generations before they were intercrossed. Genomic DNA was obtained from blood samples, and polymerase chain reaction (PCR) was used to identify mice homozygous for the IL-4-/- and IL-10-/- phenotypes.
All mice were fed standard rodent chow (Ralston Purina Co., St. Louis, MO, USA) and water ad libitum. To ensure that the environment was pathogen free, sentinel mice were routinely tested for hepatitis and Sendai viruses. All animals were kept until the age of 7 to 10 weeks before being used for experiments, which were conducted in accordance with approved IACUC protocols.
CII was dissolved in 10 mM acetic acid at a concentration of 4 mg/ml and emulsified with an equal volume of CFA containing 4 mg/ml of Mycobacterium tuberculosis strain H37 Ra (Difco Microbiology Products, Becton Dickinson, Franklin Lakes, NJ, USA) . For the induction of arthritis, each mouse received 100 μg of CII emulsified in CFA subcutaneously at the base of the tail. For other immunizations, each mouse received subcutaneously 100 μg of either peptide A12 or Ova emulsified with CFA.
The severity of arthritis were determined by visually examining each forepaw and hindpaw and scoring them on a scale of 0 to 4, as described previously . Scoring was conducted by two examiners, one of whom was unaware of the identity of the treatment groups. Each mouse was scored thrice weekly, beginning 3 weeks after immunization and continuing for 8 weeks. The mean severity score (sum of the severity scores for the group on each day/total number of animals in the group) was recorded at each time point.
Soluble DR1 was purified from culture supernatants of S2 Drosophila cells previously transfected with DRB1*0101 and DRA1*0101. Both the α- and β-chains of the DR1 constructs contain murine I-E leader sequences, followed by DRα or DRβ first domains and murine I-Eα and I-Eβ second domains. The analog (CII259-273, 263F, 266D), was inserted after the third residue of the β-chain, and a flexible ((Gly)4-Ser)3 linker was added to allow the peptide to fold into the binding groove of the DR molecule. The sequences encoding the cytoplasmic and transmembrane portions of the molecules were deleted from the cDNA by PCR, and a new stop codon was inserted at the 3' end of the second domain of the α-chain. The β-chains were altered by adding a linker and a biotinylation site 3' to the Eβ second domain, to allow site-specific addition of biotin and tetramerization by using streptavidin. The resulting cDNA was cloned into the Drosophila expression vector pRmHA-3 (gift from Dr. D. Zaller, Merck, Rahway, NJ, USA). S2 cells were transfected with a 10:10:1 ratio of chimeric DRB1 and DRA1 to pUChsNeo by using calcium phosphate precipitation. Soluble DR production was induced by 1 mM CuSO4, and 5 days later, the culture supernatant was collected and adjusted to 0.05% octyl glucoside (OcG). The DR molecules were purified by passage of the culture supernatant over an affinity column coupled with the anti-DR Ab LB3.1. The column was washed with 0.05% OcG and 0.15 M NaCl in phosphate buffer, pH 7.5, followed by 0.05% OcG and 0.5 M NaCl in phosphate buffer, pH 7.5, and a final wash with 10 mM Tris in 0.5 M NaCl, pH 7.5. The DR molecules were eluted with 100 mM Tris and 0.5 M NaCl, pH 11.2, and the fractions were immediately neutralized with acetic acid. The DR recovered was concentrated by using an Amicon Stirred Cell, quantitated by OD280 absorption and ELISA (PathScan Total DDR1 Sandwich ELISA Kit; Cell Signaling, Danvers, MA, USA), and was evaluated with SDS-PAGE before use. Biotinylation of the proteins was performed with BirA (Avidity, Denver, CO, USA) and incorporated into saturated complexes with phycoerythrin (PE)-streptavidin (BioSource International, Camarillo, CA, USA) before use.
For tetramer staining of T cells, lymph node cells were diluted to a concentration of 2.5 × 107/ml in DMEM medium (Gibco) supplemented with 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate, 0.05 mM 2-ME, 2 mM L-glutamine, and 0.1% BSA, and T-hybridoma cells were diluted to a concentration of 2.5 × 106/ml in DMEM complete. One million lymph node cells or 105 T-hybridoma cells were then aliquoted into a 96-well plate and were incubated with 1 μg of tetramer in 10 μl of complete medium supplemented with 5 mM NaN3. Cells were incubated at 37°C for 2.5 hours, at which time, antibodies to cell-surface markers were added, and cells were incubated for an additional 30 minutes at 4°C. After incubation with Abs to cell-surface markers, samples were washed 3 times with 200 μl of PBS supplemented with 0.1% NaN3 and 2% FBS, resuspended in 200 μl of PBS supplemented with 0.1% NaN3 and 2% FBS, and analyzed with flow cytometry by using a FACSCalibur (BD Biosciences). In some experiments, the draining lymph nodes of DR1 mice were harvested 10 days after immunization with CII and were incubated with tetramer and subjected to magnetic sorting to obtain a purified population of tetramer-positive cells (Miltenyi Biotec).
To determine the frequency of individual Vβ subfamilies expressed by tetramer+ T cells in DR1 mice, draining inguinal cells from DR1 mice immunized 10 days earlier with A12/CFA were incubated with tetramer labeled with PE, anti-CD4-APC, anti-α/βTCR-PerCP (BD Biosciences, San Jose, CA, USA), and an anti-TCR-Vβ-specific Ab conjugated with FITC for 30 minutes at 4°C (Mouse Vβ TCR Screening Panel; BD Biosciences). Labeled cells were washed with PBS, and a minimum of 10,000 cells was analyzed from each sample with flow cytometry by using a FACSCalibur or LSR II (BD Biosciences). The final analysis was performed by using FlowJo software (Tree Star, Ashland, OR, USA).
Mice were bled at 6 weeks after immunization, and sera were analyzed for antibodies reactive with native CII by using a modification of an enzyme-linked immunoassay (ELISA) previously described . Serial dilutions of a standard serum were added to each plate. From these values, a standard curve was derived by computer analysis by using a four-parameter logistic curve. Results are reported as units of activity, derived by comparison of test sera with the curve derived from the standard serum, which was arbitrarily defined as having 50 units of activity. Reactivity to CII was not detected in sera obtained from normal mice.
Inguinal lymph nodes from immunized mice were isolated, and the phenotype induced by A12 was determined by multiparameter flow cytometry with an LSRII flow cytometer (BD Biosciences). Cells were cultured with fluorochrome-labeled antibodies specific for CD4, CD25, or CD44 (BD Biosciences) as well as tetramer. In some experiments, intracellular labeling was performed by using antibodies specific for FoxP3 (FJK-16s; eBioscience, San Diego, CA, USA), per the manufacturer's protocol. In the experiments involving intracellular staining, specific gating was performed on both the CD4+ and the Vβ8+, Vβ14+ population of cells.
In transfer experiments, inguinal lymph node cells from DR1 mice previously immunized with the A12 analog peptide in CFA were collected 8 days after immunization, and various cell subsets were fractionated by using PE-labeled tetramers and ferromagnetic beads (Miltenyi Biotec, Gladbach, Germany), according to the manufacturer's protocol. The purity of each cell population was confirmed by flow cytometry to have > 95% purity. Recipient mice were given cells intravenously by using two protocols (a) 2 × 105 either tetramer+ or tetramer- T cell or (b) 5 × 105 of VB8+, VB14+ T cells. All recipient mice were immunized with CII either on the day of the cell transfer (prevention protocol) or 25 days before cell transfer (therapy protocol) and observed for arthritis.
To measure cytokines, inguinal CD4+ lymph node cells or purified tetramer+ T cells were cultured (5 × 105 CD4+ T cells/ml) with wild-type APCs (DR1-positive splenocytes) (1:2 ratio), which had been prepulsed with 100 μg/ml of the various peptides (A2 or A12). The CD4+ cells were collected 8 days after immunization (with either OVA, CII, or A12 emulsified in CFA) by negative selection by using ferromagnetic beads, according to the manufacturer's protocol (Miltinyi Biotech). (Each cell population was confirmed by flow cytometry to have > 95% purity). Each culture was set up by using pooled cells from three mice run in triplicates, and supernatants were collected at 72 hours and analyzed for the presence of IL-4, IL-10, IL-2, INF-γ, and IL-17 by using a Bio-plex mouse cytokine assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Values are expressed as picograms per milliliter and represent the mean values for each group taken from three separate experiments.
Mean severity scores, antibody titers, and cytokine levels were compared by using the Mann-Whitney test.
We previously reported that the A12-induced suppression of arthritis is achieved primarily by T cells . To identify and characterize clearly the cells responsible for this effect, we generated DR1 multimers (tetramers) that have a covalently linked collagen analog, so that the A12 peptide occupies the majority of the MHC molecule binding clefts [9–11]. Biotinylated DR1-A12 molecules were generated and assembled into tetramers by using phycoerythrin-streptavidin. To determine the binding specificity of our tetramer, we used a well-characterized panel of DR1-restricted CII-specific T-cell hybridomas [8, 14] and cultured them with the A12-tetramer before analysis with flow cytometry. As shown in Figure 1, the A12-tetramer bound only to the collagen-specific DR1 restricted hybridomas (example, DR1-E174, middle panel, Figure 1, which is representative of nine individual CII-specific hybridomas (mean fluorescence (MF) = 11,400 ± 392). Conversely, the tetramer was antigen specific, unable to recognize DR1-restricted hybridomas that are specific for hemagglutinin (example, DR1.HA.8; right panel, Figure 1, a DR-1 restricted hybridoma that recognized HA, MF = 493 ± 256; n = 4). Finally, we determined that the tetramer binding was MHC specific, incapable of binding collagen-specific hybridomas that were restricted by another MHC (for example, DR4-restricted collagen-specific hybridomas, MF = 462 ± 280, n = 4, P < 0.0001; compared with the MF of 11,400 ± 392 of the DR1-restricted CII-specific hybridomas, n = 9). These data confirm that that our A12/DR1-tetramer recognizes only the collagen-specific, DR1-restricted T cells in our panel.
Functional analysis and specificity of the recombinant DR1-A12. Tetrameric DR1-A12 was tested for its ability to bind specifically to DR1-restricted, CII-specific T cells. A panel of T-cell hybridomas was cultured with the tetramers in saturating concentrations at 37°C for 3 hours. An anti-CD4 antibody was added in the last 30 minutes before the cells were washed and analyzed on the flow cytometer. The left panel demonstrates an unstained control. The middle panel shows hybridoma DR1.E174, which is representative of tests performed on nine individual CII-specific DR1-restricted hybridomas. The right panel (DR1.HA.8) is representative of four control T-cell hybridomas specific for an HA peptide in the context of DR1. Previous testing has shown that none of these hybridomas is bound by the irrelevant tetramer DR1-HA, which contains the immunodominant determinant of hemagglutinin ). These data confirm that that our A12/DR1-tetramer recognizes only the collagen-specific, DR1-restricted T cells in our panel.
Although the concept of "suppressor" T cells was first put forward 40 years ago , an increasing number of immune cell populations have been reported to play a role in the regulation of autoimmunity . Therefore, it is important to characterize precisely the cells induced by the analog peptide A12. We chose to search for the A12-specific T cells directly ex vivo. DR1 mice were immunized with A12 or Ova (as a control), and 10 days later, T cells from inguinal lymph nodes were stained with DR1-A12 tetramer and anti-CD3, either immediately or after 4 days of restimulation in culture with the A12 peptide. As shown in Figure 2, an increase in the percentage and number of CD3+ T cells that bound the DR1-A12 tetramer could be observed both ex vivo and after stimulation in culture compared with controls. Although the ex vivo analyses of DR1-restricted, A12-specific T cells revealed only a small population of tetramer+/CD3+ T cells (1.5% ± 0.3% versus 0.5% ± 0.3%; P ≤ 0.05; n = 6), by day 4 of stimulation in culture, a clearly discernible population (8.5% ± 0.5% versus 0.5% ± 0.4%; P ≤ 0.05; n = 6), representing a fivefold expansion of cells, was visible. The specificity of the tetramer staining was quite clear, because nonspecific binding (cells derived from DR1 mice that had been immunized with Ova, but stained with a DR1/A12 peptide tetramer) was extremely low (Figure 2b, d).
Ex vivo analysis of the development of the A12-specific T-cell response in DR1 Tg mice. DR1 mice were immunized with either A12 or OVA, and individual mice were killed on day 10 after immunization. Cells from inguinal lymph nodes were collected and either analyzed immediately (a, b) or stimulated for 4 days with the A12 peptide or Ova before analysis (c, d). (Lower panel) DR1 mice were immunized with either CII/CFA (e) or Ova (f) and, at the time of onset of arthritis, were treated subcutaneously (100 μg/mouse in IFA) with either A12 peptide (e) or Ova (right) (f). Cells were collected from inguinal lymph nodes on day 26 after treatment with peptide, and the cells were analyzed for the presence of tetramer-positive cells directly ex vivo with flow cytometry. All cells were analyzed for the presence of A12-specific T cells by using the DR1-A12 tetramer and anti-CD3 or anti-CD4, anti-CD8, and anti-CD19. Anti-CD8 and anti-CD19 were used simultaneously in the FL4 channel, and anti-CD3 or anti-CD4 was used in the FL1 channel. The cells represented are CD19-/CD8-/CD3+ or CD19-/CD8-/CD4+. Data are representative of at least six mice per time point and are based on a minimum of 100,000 cells analyzed.
In clinical settings, analog peptides will be administered therapeutically during episodes of active arthritis. To characterize the A12-specific T-cell response that develops within an inflammatory milieu, DR1 mice were immunized with CII/CFA, and once arthritis was clearly established, the mice were treated subcutaneously with A12 peptide. When the DR1-A12 multimers were used to stain inguinal lymph node cells from arthritic mice evaluated 26 days after peptide treatment, a distinct population of tetramer+ cells was detected in the A12-treated mice (1.2% ± 0.5% versus 0.2% ± 0.3%; P ≤ 0.05; n = 6; Figure 2e). Again the specificity of the tetramer staining was clear, because nonspecific tetramer binding by cells derived from DR1 mice that had been immunized with Ova remained low (Figure 2f).
Although we previously reported that CII-specific T cells restricted by DR1 are clearly oligoclonal , the degree of heterogeneity among analog A12-responding T cells remained unclear. Therefore, we analyzed the T cells that migrated to the inguinal lymph nodes after immunization with A12 and analyzed them for TCR β-chain variability. As shown in Table 1, when cells were analyzed by specifically gating on individual Vβ populations after co-staining with tetramer, two Vβ types were explicitly preferred, Vβ8 (Vβ8.1, Vβ8.2, or Vβ8.3) and Vβ14. A significant tetramer+ T-cell population was detected in each of these two subsets (Vβ8 and Vβ14), whereas all other Vβ antibodies tested had tetramer levels indistinguishable from background. Collectively, Vβ14 and Vβ8 accounted for > 97% of the tetramer+ T cells, thus identifying them as the major T-cell population responding to the A12 analog determinant presented by DR1. The tetramer-negative cells had a T-cell repertoire comparable to that of wild-type DR1 mice, which we previously reported .
To determine the frequency of individual Vb subfamilies expressed by tetramer+ T cells in DR1 mice, draining inguinal cells from DR1 mice immunized 10 days earlier with A12/CFA were incubated with tetramer labeled with PE, anti-CD4-APC, anti-a/bTCR-PerCP, and an anti-TCR-Vβ-specific Ab conjugated with FITC. Labeled cells were analyzed from each sample with flow cytometry, and the final analysis was performed by using FlowJo software. Data shown represent the mean values from three separate analyses. Virtually all the tetramer+ T cells expressed either the Vβ14 or the Vβ8 (Vβ8.1, Vβ8.2, or Vβ8.3) TCR segment, identifying them as the major T-cell population responding to the A12 peptide presented by DR1.
Our next approach was to characterize the phenotype of the heterogeneous CD4+ T-cell subset generated by interaction with A12 by using multiparameter flow cytometry. Naive T cells activated by antigen/APC interaction are expected to expand and differentiate into effector T cells, forming subpopulations that express activation markers, such as CD25, and memory markers, such as CD44. We previously showed that T cells recognizing the immunodominant T-cell determinant of CII upregulate both activation and memory markers on stimulation with CII [7, 17]. However, when A12-tetramer+ cells were tested for the presence of activation and memory markers, their expression (CD25 and CD44) was undetectable either directly ex vivo or after 4 days of culture (data not shown). Similarly, the numbers of Tregs (identified as Foxp3+ and CD25+; Figure 3) remain at background levels in populations of CD4+, VB8+, and VB14+ T cells that have been immunized with A12/CFA. Taken together, our data reveal that a population of A12-specific inhibitory T cells migrates to the inguinal lymph nodes after immunization with A12/CFA and develops a distinct oligoclonal TCR response without memory, activation, or Treg-specific identifiers.
A12-responsive T cells are not Tregs. Inguinal lymph node cells from DR1 Tg mice immunized 10 days earlier with A12/CFA (left panel), A2/CFA (middle panel), or Ova/CFA (right panel) were cultured directly ex vivo with anti CD25, CD4+, VB8+, VB14+, and intracellular FoxP3. Selective gating was performed on the CD4+, VB8+VB14+ cells. Before intracellular staining, a sample of the A12-specific cells was stained with DR1-A12 tetramer to confirm that the VB8+, VB14+ population was enriched for tetramer+ cells (15% tetramer+). The experiments were repeated after 4 days of culture with the A12 peptide in vitro, and the results were similar to those shown. These data indicate that the numbers of Foxp3+, CD25+ Treg cells remain at background levels after immunization with A12/CFA.
A major mechanism by which T cells function is through the secretion of cytokines, either inflammatory (Th1, Th17) or suppressive (Th2). To test for cytokine secretion, cells from the inguinal lymph nodes of mice previously immunized with A12/CFA were fractionated with ferromagnetic beads into tetramer+ and tetramer- subsets and cultured with APCs pulsed with the immunodominant collagen peptide. Control T cells were purified from mice immunized with either CII or Ova. As shown in Figure 4, supernatants from tetramer+ cell cultures contained predominantly Th2/suppressive cytokines (IL-4 and IL-10) in response to collagen, whereas the tetramer-negative population did not react. Conversely, control A2-specific T cells (from CII-immunized mice) secreted both suppressive (IL-4 and IL-10) and inflammatory (IFN-γ and IL-17) cytokines to collagen, whereas Ova-responsive cells were generally nonreactive. These data make it clear that tetramer+ cells are responsible for the suppressive cytokine secretion after activation by A12. Very little evidence suggests that Tregs, NKT cells, or any other potential suppressive subsets play a major role.
Cytokine responses to A12-tetramer + cells. DR1 mice were immunized with Ova (open bars), CII (solid bars), or A12 (hashed and striped bars), and the draining inguinal lymph node cells were selected to obtain the CD4+ populations. The A12-immunized lymph node cells were further fractionated into tetramer+ (left hashed bars) and tetramer- (striped bars) populations. The cells were cultured (5 × 105 CD4+ T cells/ml) with wild-type APC (DR1-positive splenocytes; 1:2 ratio) prepulsed in vitro with the immunodominant collagen peptide. Supernatants were collected and tested for the presence of each cytokine by using a multiplexed ELISA. The supernatants were collected from Ova-specific CD4+ T cells (open bars), CII-specific T cells (solid bars), DR1-A12 tetramer+ T cells (left hashed bars), or tetramer- T cells (striped bars). Results shown represent the mean ± SD of three separate experiments and are expressed in picograms per milliliter of culture supernatants. The Th1/Th2 ratio was 13:1 in T cells responding to CII, whereas the Th1/Th2 ration was 0.5:1 in the DR1-A12 tetramer+ T cells.
To date, our studies of A12-tetramer+ T cells have focused primarily on T cells detected in inguinal lymph nodes after immunization with A12/CFA. Interestingly, when T cells from inguinal lymph nodes of cells immunized with CII/CFA (without treatment with analog) were collected 10 days after immunization and stained for the presence of A12-tetramer+ T cells, we detected a small population (0.86 ± 0.2 A12-tetramer+ T cells compared with 0.23 ± 0.04 tetramer+ T cells from Ova immunized mice (P ≤ 0.007 from three separate analyses). Although more work must be done to characterize definitively the cytokine profiles and phenotypes of these cells, we have found that T cells collected from lymph nodes after immunization with CII/CFA secrete suppressive cytokines (primarily IL-4 and IL-10) in response to A12/DR1 and not inflammatory cytokines . These data suggest that activated T cells can be redirected to respond to the analog with a suppressive cytokine profile and that this redirection can occur in a setting of active inflammation. Moreover, we have found that the cytokine response to the analog A12 remains suppressive, regardless of which routes were used for administration or whether the peptide was given before or after immunization .
We previously showed that IL-4 can attenuate arthritis. However, it remained unclear whether other cytokines affect the A12 mechanism of action. To confirm and compare the importance of other Th2 cytokines in vivo, we developed DR1 mice that were genetically deficient in IL-4 and IL-10, so that groups of 10 IL-4-/- or IL-10-/- mice could be treated with A12 at the time of immunization with CII/CFA and compared with wild-type DR1 mice for the severity of the development of arthritis. As shown in Figure 5, the cytokine IL-10 appears to play a partial role in the A12 prevention of arthritis (Figure 5), whereas IL-4 is more potent. Interestingly, T cells from A12-treated IL-10-/- mice produced IL-4 when cultured with collagen (IL-4 = 76 ± 21 pg/ml), but the levels were not different from those produced by A12-treated IL-10+/+ T cells (IL-4 = 65 ± 14 pg/ml). These data extend our previous observation to confirm that the effectiveness of A12 in treating arthritis is mediated at least in part by both IL-4 and IL-10.
Analog peptides in IL4 -/- and IL10 -/- mice. Groups of DR1+ IL-4-/- , DR1 IL4+/+ littermates, DR1 IL10-/-, and DR1 IL10+/+ littermate mice were immunized with CII/CFA. Mice were treated with either the A12 peptide or Ova (0.6 mg) subcutaneously in IFA at the time of immunization. The mean severity scores for arthritis are shown at various times after immunization. The DR1 IL4+/+ mice treated with A12 differ significantly from DR1 IL4+/+ mice treated with Ova (P < 0.0005 on day 53). Conversely, the DR1 IL4-/- group treated with A12 did not differ from Ova-treated controls. Data shown are representative of four separate experiments. Similarly, the DR1 IL10+/+ mice treated with A12 were significantly suppressed when compared with the DR1 IL10+/+ mice treated with Ova, P < 0.001 on day 53). Although the DR1 IL10-/- mice had suppression of arthritis compared with Ova-treated controls (P ≤ 0.05), the suppression was not as potent as that obtained with DR1 IL10+/+ mice. Similarly, the incidence of arthritis was 15% in IL10+/+ A12-treated mice, which was significantly different from that of OVA-treated IL10+/+ mice (67%; P = 0.004). Conversely, the A12-treated IL10-/- mice had an arthritis incidence of 36%, which was not statistically different from the IL10+/+ Ova-treated mice. Data shown are representative of three separate experiments.
To resolve whether tetramer-positive T cells could successfully suppress arthritis in the CIA model, we performed passive transfer experiments . Draining inguinal lymph node cells from DR1/TCR Tg mice previously immunized with A12/CFA were collected, and tetramer+ cells were fractionated by using ferromagnetic beads. Either tetramer+ cells or tetramer- controls were infused intravenously into DR1 tg mice, by using a (prevention protocol) so that mice could be observed for effects on collagen-induced arthritis. When infused before the induction of arthritis, we observed (Figure 6A), that the tetramer+ population of A12-immune T cells was extremely effective in suppressing arthritis, compared with tetramer-negative control LN cells.
Cell transfer: prevention of arthritis. Tetramer+ T cells from draining lymph nodes were collected from DR1Tg mice immunized 10 days earlier with A12/CFA. DR1 mice (n = 3) were infused with 2 × 105 cells of either tetramer+ or tetramer-, A12-primed CD4+ T cells. All recipients were immunized with CII/CFA on the day of the cell transfer and observed for arthritis. (A) Mean severity scores over time. As indicated, only the tetramer+ A12-immune cells could prevent collagen-induced arthritis (final severity scores comparing square versus triangle of 1.3 ± 2.5 versus 5.4 ± 3.2; P ≤ 0.01. The cell-transfer experiments have been repeated with both unfractionated cells and VB8+, VB14+ purified T cells from A12-immunized lymph nodes with similar results. Suppression of established arthritis (B). Inguinal lymph node cells from A12-immunized DR1 mice were fractionated into a VB8, VB14+ population and a VB8, VB14- population. DR1 mice (n = 10) were immunized with CII/CFA and observed for the development of arthritis. On day 23 after immunization, when arthritis was established, each mouse was infused intravenously with 5 × 105 cells of either VB8, VB14+ T cells (enriched for 7.5 × 104 tetramer+ cells) or VB8, VB14- T cells. (B) Mean severity scores over time. As shown, only the VB8, VB14+ A12-immune T cells could suppress active arthritis in the collagen-induced arthritis model (final severity scores comparing (triangle versus circle) of 1.5 ± 1.0 versus 5.6 ± 1.6; P ≤ 0.03). Data shown are representative of three separate experiments.
With a different strategy to enrich for A12-responsive T cells, we collected Vβ8 and Vβ14 populations from A12-immunized lymph nodes by means of ferromagnetic beads and subset-specific antibodies for positive selection. Flow cytometry confirmed that this population was highly enriched for the tetramer+ T cells before infusion into mice. When these cells were given to mice after arthritis had been established (Figure 6B), arthritis was significantly attenuated. Mean antibody titers to CII taken 6 weeks after immunization were decreased in mice given the Vβ8-, Vβ14- enriched T cells, compared with mice given Vβ8, Vβ14- CD4+ T cells (Table 2).
Inguinal lymph node cells from A12-immunized DR1 mice were fractionated into a Vβ8, Vβ14+ population and a Vβ8, Vβ14- population by using ferromagnetic beads. DR1 mice (n = 10 for each group) were immunized with CII/CFA, and on day 23 after immunization, when arthritis was established, each mouse was infused intravenously with 5 × 105 cells of either Vβ8, Vβ14+ T cells or Vβ8, Vβ14- T cells. To confirm the importance of the cytokine responses after cell transfer in vivo, inguinal lymph node cells were taken from the mice 28 days after treatment and were cultured with murine collagen so that supernatants could be analyzed for the presence of cytokines. When cultures from mice treated with the Vβ8+, Vβ14+ cells were compared with those from mice treated with the Vβ8-, Vβ14- cells, the IL-4 response to murine collagen was elevated, whereas the inflammatory cytokines (IFN-γ and IL-17), were suppressed. Mean antibody titers to CII in sera taken 6 weeks after immunization were similarly suppressed in mice given the Vβ8, Vβ14+, enriched T cells, compared with mice given Vβ8, Vβ14- CD4+ T cells.
To confirm the effectiveness of tetramer+ T cells in vivo, inguinal lymph node cells were taken from the mice 28 days after treatment and were cultured with collagen so that supernatants could be analyzed. When cultures from mice treated with the Vβ8+, Vβ14+ cells were tested for cytokines, the IL-4 response to murine collagen was increased, whereas the levels of the inflammatory cytokines were decreased when compared with those from mice treated with the Vβ8-, Vβ14- cells (Table 2). Collectively, the in vivo and in vitro experiments confirm that the suppression of arthritis by A12-specific T cells involves active secretion of suppressive cytokines (specifically IL-4 and IL-10), resulting in a downregulation in the inflammatory cytokines IL-17 and IFN-γ.
The development of class II MHC tetramers has been more challenging than that of class I tetramers, in part because, unlike class I (which can be assembled from HLA heavy chain, and excess peptide and β2-microglobulin), the class II molecule is an assembly of α and β chains, both of which are required for peptide binding. Proper folding is required for the formation of a stable complex. The most successful strategy involves producing empty class II molecules that can be loaded with exogenous peptide and assembled as MHC tetramers. However, A12 has an extremely low binding avidity for the MHC, making that approach unlikely to be successful. In this study, we developed a highly specific HLA-DR1-A12 tetramer in which we overcame the low-avidity problem by covalently linking the peptide to the DR1. This strategy allows successful identification of A12-specific T cells in the wild-type mouse.
Ex vivo studies revealed the expansion of A12-specific T cells in the inguinal lymph nodes after treatment with the peptide. Moreover, we showed that DR1-A12 tetramer+ T cells have significant antigen-specific characteristics, including a limited TCR repertoire, and the expression of high levels of Th2/suppressive cytokines despite constituting only 1.5% or less of the CD4+ population in vivo. The population of A12-specific T cells can be expanded in vitro to increase fivefold after 4 days of culture. Although the numbers of A12-specific T cells may be small in terms of the total CD4+ T-cell population, this still represents at least a 1,000-fold expansion of these cells, given an estimated antigen-specific T-cell precursor frequency of 105 to 106. In all, this approach demonstrates that a detailed ex vivo image of the developing T-cell response to an analog peptide can be achieved by using class II tetramers, and our data suggest that analog-specific T cells can expand in vivo and remain detectable in draining lymph nodes for a significant period.
The T cells identified by the A12-CII tetramer expressed primarily Th2/suppressive cytokines (IL-4 and IL-10) that are associated with autoimmune suppression. In comparison, control cells specific for collagen secreted significant amounts of all subclasses of cytokines, both inflammatory (IFN-γ, IL-17) and noninflammatory (IL-4 and IL-10). Both IL-10 and IL-4 are known to suppress CIA when they are administered to mice [19–22]. By using mice genetically deficient in either IL-4 or IL-10 that have been treated with the A12 peptide, we now extend our previous studies to demonstrate that both cytokines play an important role in the function of A12, IL-4 > IL-10. Our previous work is consistent with the concept that Th2/suppressive cytokines have a unique role in the suppression of arthritis . These elevated levels of Th2 cytokines in the tetramer+ T cells support their identification as A12-specific T cells. They probably play a significant role in promoting the development of suppression in this arthritis model.
This tetramer-based approach to the analysis of an autoimmune T-cell response offers several advantages over conventional approaches. First, cells are studied ex vivo, thus avoiding any tissue-culture effects on gene expression and T-cell function. Stable tetramer binding to T cells can be achieved within 2 hours, and the assay is performed at physiologic temperatures. Second, all measurements of phenotype and function are focused on T cells of the specificity of interest instead of an averaged analysis of the total T population derived from the lymphoid tissue. Although the frequency of the Ag-specific CD4+ T cells of interest is low both in our analyses and in those of others , this problem can be overcome by analysis of large numbers of cells and by focusing the analysis on specific subpopulations. In terms of data acquisition and analysis, we found that care must be taken in quantitating these low-frequency events, because background or nonspecific fluorescence can significantly compete with a 1% or less positive population. The use of both inclusion (CD3+ or CD4+) and exclusion (CD8-/CD19-) gates for data analysis helped to ensure the high degree of sensitivity and reproducibility in identifying these low-percentage events.
Although the A12-specific T-cell response could be clearly visualized by our DR-A12 tetramers, some questions remain as to whether these tetramers detect all the DR1-restricted CD4+ T cells specific for the A12 peptide. Although the frequency of A12-specific T cells we observed is similar to that reported in other MHC class II tetramer-based studies of Ag-specific T cells, it is still possible that we are failing to identify some small population of CII-specific T cells that might have low affinity for DR1-A12 or for some other unknown reason fail to bind the tetramer. Despite these concerns, however, this tetramer-based approach has significantly broadened our understanding of the role and function of analog-specific T cells in wild-type mice. The detection of A12-specific T cells in the inguinal lymph nodes of arthritic animals after treatment with peptide and the discovery that tetramer+ T cells can suppress arthritis when infused into arthritic mice indicates that these cells may play a direct role in altering the course of autoimmune disease.
The success of the altered peptide ligand, glatiramer acetate (Copaxone), in treating patients with multiple sclerosis (MS), suggests that peptide therapies based on naturally occurring proteins may provide an effective alternative for the treatment of autoimmune arthritis. Copaxone is a mixture of peptides composed of four randomly arranged amino acids (L-alanine, L-lysine, L-glutamic acid, and L-tyrosine), whose chemical structure resembles that of the myelin basic protein molecule . The mechanism of action of successful APLs appears to be the induction of inhibitory T cells, so that inactivated T cells become Th2/inhibitory cells (which secrete antiinflammatory cytokines) instead of Th1 and Th17 cells (which secrete proinflammatory cytokines), a phenomenon known as "immune deviation." The effectiveness of A12 is based on its ability to transform both potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells . Moreover, this deviation also downregulates other proinflammatory Th1 and Th17 cells, regardless of their antigenic specificity, in a process known as "bystander suppression." APLs are potentially safer than existing therapies because they contain a modification of an endogenous naturally occurring protein, used to arrest the autoimmune T-cell attack and allow tissue repair.
We demonstrated that peptide/MHC multimers can be used to detect inhibitory cells induced by altered peptide ligands, even when the peptides have extremely low avidity for the MHC. These cells are highly effective in suppressing autoimmune arthritis. These data demonstrate that analog-specific multimers are powerful tools for detecting inhibitory T cells, useful for studies of human T cells in upcoming clinical trials for RA. We anticipate that future studies will outline the differences between A12-specific T cells from healthy control subjects and patients with RA.
This work was supported, in part, by USPHS grants AR-55661, AR-55266, and program-directed funds from the Department of Veterans Affairs and the Arthritis Foundation.
All authors made substantial contributions to the conception and design of the experiments. MK and DLC designed and carried out all the studies analyzing the T cells in vitro; EFR designed and developed the tetramers; MLB and DDB designed and participated in the studies using flow cytometry; LKM, JS, and AHK designed and participated in studies using the collagen-induced arthritis animal model. All authors were involved in drafting and revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.


You are here: Home / Main Dish / Chicken / Slow Cooker Hawaiian BBQ Chicken Sandwiches – an easy twist on an old favorite!
Does it get any easier than tossing chicken in the slow cooker with some barbecue sauce? It's a classic and a family favorite. This recipe is really more of a twist on serving the barbecue chicken. You start with those same two ingredients, but the sandwiches get bumped up a notch with the addition of pineapple slices and cheese.
I had intended to make another recipe I have for Hawaiian BBQ Sandwiches. That recipe uses grilled chicken and ham. It's not hard, but I was feeling super lazy. Plus, I'm loving using the slow cooker lately!
So I cooked the chicken with our favorite barbecue sauce in the slow cooker. At dinner time, I shredded the chicken and served it on toasted buns, topped with sliced pineapple and provolone. A quick turn under the broiler and they were ready to eat.
Oh my goodness! The pineapple and provolone really set these sandwiches apart from plain bbq chicken sandwiches. It added so much flavor to the meal and it's an easy step.
Put the chicken and bbq sauce in the slow cooker.
Place meat on buns. Top with pineapple and cheese.
These look delicious Tiffany ~ love that you came up with a new way to use simple crockpot barbecue chicken!
Did you have the sandwiches open or closed when you broiled them?
I broiled them open. If the top bun is looking too toasty, just take it off the baking sheet before you broil the sandwiches.
OH.MY.GOODNESS Tiffany! This sammy looks absolutely heavenly. My hubs is not wild about BBQ, but I am and will have to try these soon. Thank you so much for sharing. YUUUMMMM!
I love using my crockpot, and this recipe sounds easy!
I made this recipe today with just a small twist in that I made my own B-B-Q sauce. It was spiced up a bit and I rolled the pulled BBQ chicken into corn tortillas and fried them in Avacado oil. Top with chopped lettuce, onion, and fresh tomato, and you choice of taco sauce. It was SUPER!
I'm making these today…can't wait for dinnertime! YUMMM!!
When do i add the pineapple????
You just use the pineapple as topping for the sandwiches. Add it when you assemble them.
Use Sweet Baby Ray's "Honey Chipolte" BBQ Sauce. My favorite esp with chicken or pork in the Crock Pot!
Maybe a situpid question- but is this recipe time For frozen or thawed chicken breasts? Thanks in advance!
I've done it either way. If I'm using frozen, I start the crockpot on high.
These are outstanding! We never eat plain bbq chicken sandwiches anymore!
I am gone for 12 hours on workdays, is that too long to leave it in the slow cooker, on low?
That will work great for this recipe. Just be sure your crock is pretty full, otherwise it will cook too hot.
That sounds fantastic to me!
Looks delicious and seems so easy! Also love the touch of Polish Pottery as your serving piece, nice touch!


Former Google Engineers Launch Search Engine – WSJ.com.
This is an interesting article about a new search engine called Cuil, which I had seen on Twitter a couple of days ago. It will be interesting to watch and see whether it can make a dent in Google's dominance.
Update: I originally posted this using the WordPress.com Press This bookmarklet. It will be interesting to experiment with that, and I'll report on it later.
Author Lee AasePosted on July 29, 2008 Categories Internet, News MediaTags Cuil, Google, Search, search engines2 Comments on Can Cuil Cut it?
I'm at the last session of Media Relations 2008 in San Francisco, attending a presentation by Brian Kaminski of iProspect, Lee Odden of TopRank Online Marketing and Dr. David Hulme, Vision Media.
Note: Lee did an excellent review of Mike Moran's keynote from today. He also says he's going to put his presentation on Slideshare, so I will link to it when it's up.


text:
Q: Handle key events Ctrl+Tab and Ctrl+Shift+Tab I want to handle the two key events Ctrl+Tab and Ctrl+Shift+Tab in order to switch between tabs in my application ("forward" and "backward" respectively). However, this doesn't seem to work as expected.
This is my current code (minimal example):
import QtQuick 1.1

Item {
    width: 100
    height: 100

    focus: true

    Keys.onPressed: {
        if(event.modifiers & Qt.ControlModifier) {
            if(event.key === Qt.Key_Tab) {
                if(event.modifiers & Qt.ShiftModifier)
                    console.log('backward')
                else
                    console.log('forward')
            }
        }
    }
}

I ran this piece of code with qmlviewer (Qt version 4.8.2)
Output when pressing Ctrl+Tab:
forward
forward

Output when pressing Ctrl+Shift+Tab:

none

So I see two errors: The former key sequence is handled twice while the other one not at all.

*

*EDIT: The reason that the other one doesn't get handled at all is solved, see comments.

Why does this happen and how can I solve this?
Note: I already use Qt Components for Desktop in my application, so it's OK if you know a solution requiring this module.

A: You have to accept the event, otherwise the event is propagated to the parents until it is accepted. The following code worked for me.
Item {
    width: 100
    height: 100

    focus: true

    Keys.onPressed: {
        if(event.modifiers && Qt.ControlModifier) {
            if(event.key === Qt.Key_Tab) {
                console.log('forward')
                event.accepted = true;
            }
            else if(event.key === Qt.Key_Backtab) {
                console.log('backward')
                event.accepted = true;
            }
        }
    }
}

Edit: This behavior lets parents handle events that the child could not, for things like hotkeys.
Hope this helps!

redpajama_set_name: RedPajamaStackExchange


text:
Q: When selecting row 0 in JTable the selection handler goes mad I'm having problems with the selection handler for a JTable.
The table is updated every 15th second from a refresh thread in the Control.
I want to select a row in my JTable and extract a column contents that I will use to build a filename from.
As long as row 0 is not selected during a refresh everything works ok. But if row 0 is selected when the refresh is triggered it looks like it jumps between the setAppserverData procedure and the event handler until it exceeds the rowCount for the table and I get an IndexOutOfBoundsException.
I'm a Java newbee and this is on the edge of my knowledge, so I'm having a hard time figuring out what is wrong with the code. My gut feeling is that it has something to do with me trying to achieve a MVC structure and I fail to separate the code in the correct way.
// The controller
// Appserver worker
class AppserverWorker extends SwingWorker<Integer, Integer>{
    protected Integer doInBackground() throws Exception{

        // Check appservers
        while(true){
            theModel.readAppservData();

            // Update action buttons
            try {
                if(!theModel.isStatusOk()){
                    theGui.actionButtonPanel.setAppServBtnColor("RED");
                    //              theGui.actionButtonPanel.setButtonFlashOn();
                }else theGui.actionButtonPanel.setAppServBtnColor("GREEN");
            } catch (IllegalArgumentException e1) {
                sysLogger.logMsg("SEVERE",e1.getMessage());
                JOptionPane.showMessageDialog(null, e1.getMessage());
            }

            // Update GUI
            theGui.extAppServPanel.setAppserverData(theModel.getAppservData());

            // Sleep refresh time
            // TODO read refresh from init table
            try {
                Thread.sleep(appServRefreshTime);
            } catch (InterruptedException e) {}
        }
    }

// List selection handler
class SharedListSelectionHandler implements ListSelectionListener {
    public void valueChanged(ListSelectionEvent e) {
        if (!e.getValueIsAdjusting()){                  // To avoid double trigger of listener
            System.out.println(">>>>>>>>>  Selection list handler >>>>>>>>");
            String fileName = null;
            String fileExt  = ".txt";
            ListSelectionModel lsm = (ListSelectionModel)e.getSource();
            try {
                int selectedRow = lsm.getAnchorSelectionIndex();                                // Get the row clicked
                if (selectedRow>=0){                                                            // If -1 no row selected
                    fileName = theGui.extAppServPanel.getAppservName(selectedRow)+fileExt;      // Get appserver name and build file name
                    theModel.readCollectFile(InitParameters.collectFilePath, fileName);         // Read collect file
                    theGui.extAppServPanel.setAppservInfo(theModel.getCollectFileContent());    // Fill the text panel with collect file
                }
            }
            catch(FileNotFoundException e1){
                sysLogger.logMsg("SEVERE",this.getClass().getSimpleName()+": Collect file "+fileName+" is missing");
                JOptionPane.showMessageDialog(null, "Collect file "+fileName+" is missing");
            }

            catch(IOException e1){
                sysLogger.logMsg("SEVERE",this.getClass().getSimpleName()+": System error: An I/O error occurred");
                System.err.println("System error: An I/O error occurred");
                System.exit(0);;
            }
        }
    }
}

// The view
public class ExtAppServPanel extends JPanel {

private JTable table;
private DefaultTableModel model;

private JTextArea textAreaInfo;
private JButton btnSaveInfo;
private List colData;

public boolean ignoreTableChanges = false;

/**
 * Constructor
 */
public ExtAppServPanel() {
    System.out.println(this.getClass().getSimpleName()+": Constructor");

    setLayout(new MigLayout("", "[350:376.00,grow,leading]", "[100px:100,grow][][48.00,grow][]"));
    this.setBorder(new TitledBorder(UIManager.getBorder("TitledBorder.border"), "Application servers info", TitledBorder.LEADING, TitledBorder.TOP, null, new Color(0, 0, 0)));

    // Set up table
    String[] columnNames = {"Server name", "Type","Status"};
    model = new DefaultTableModel(null,columnNames);
    table = new  JTable(model){

        // Color code rows
        @Override
        public Component prepareRenderer(TableCellRenderer renderer, int row, int col) {
            Component comp = super.prepareRenderer(renderer, row, col);

            if (!isRowSelected(row)){
                comp.setBackground(getBackground());
                int modelRow = convertRowIndexToModel(row);
                String type = (String)getModel().getValueAt(modelRow, 2);
                if ("FAIL".equals(type)) comp.setBackground(Color.RED);
                if ("WARNING".equals(type)) comp.setBackground(Color.YELLOW);
            }
            return comp;
        }
    };

    // Set grid
    table.setGridColor(Color.LIGHT_GRAY);

    // Set width and alignment
    table.getColumnModel().getColumn(1).setMinWidth(200);
    table.getColumnModel().getColumn(1).setMaxWidth(200);
    table.getColumnModel().getColumn(1).setPreferredWidth(200);

    table.getColumnModel().getColumn(2).setMinWidth(75);
    table.getColumnModel().getColumn(2).setMaxWidth(75);
    table.getColumnModel().getColumn(2).setPreferredWidth(75);

    // Add scrollpane
    JScrollPane scrollPane = new JScrollPane(table);
    scrollPane.setVerticalScrollBarPolicy(ScrollPaneConstants.VERTICAL_SCROLLBAR_ALWAYS);
    scrollPane.setHorizontalScrollBarPolicy(ScrollPaneConstants.HORIZONTAL_SCROLLBAR_ALWAYS);
    table.setFillsViewportHeight(true);
    this.add(scrollPane, "cell 0 0,grow");

    // Set up text area
    JLabel lblDetails = new JLabel("Details");
    this.add(lblDetails, "cell 0 1,alignx leading");

    textAreaInfo = new JTextArea();
    textAreaInfo.setBackground(Color.BLACK);
    textAreaInfo.setForeground(Color.GREEN);
    textAreaInfo.setEditable(false);
    textAreaInfo.setFont(new Font("Lucida Console",Font.PLAIN,10));

    JScrollPane scrollPane_1 = new JScrollPane();
    scrollPane_1.setViewportView(textAreaInfo);
    this.add(scrollPane_1, "cell 0 2,grow");

    // Save button
    btnSaveInfo = new JButton("Save");
    add(btnSaveInfo, "cell 0 3,alignx right");
}

/*
 * Setters and getters
 */
public void setAppserverData(ArrayList<ArrayList<String>> dataRecord) {
    ArrayList<String> dataCol = new ArrayList<String>();

    // Init values array from result set
    try {
        model.setRowCount(0);   // <<<<<<<<<<  Problem
        String[] arrayRow;
        String status = "Ok";

        // Get first row from list
        dataCol = dataRecord.get(0);
        arrayRow = new String[dataCol.size()];
        arrayRow[0] = dataCol.get(2);
        arrayRow[1] = dataCol.get(1);
        arrayRow[2] = dataCol.get(4);

        for (int i = 0; i < dataRecord.size(); i++){
            dataCol = dataRecord.get(i);

            // Check status
            if (dataCol.get(4).toUpperCase().equals("FAIL")){
                status = "FAIL";
            }else if (dataCol.get(4).toUpperCase().equals("WARNING")){
                status = "WARNING";
            }

            if (!dataCol.get(2).toUpperCase().equals(arrayRow[0].toUpperCase())){
                arrayRow[2] = status;                           // Override status
                System.out.println(">>>>>>>>>  Updating table >>>>>>>>");
                model.addRow(arrayRow);                         // Add row to table
                arrayRow = new String[dataCol.size()];
                arrayRow[0] = dataCol.get(2);
                arrayRow[1] = dataCol.get(1);
                arrayRow[2] = dataCol.get(4);
                status = "Ok";
            }
        }
        setAppservName();
    } catch (Exception e) {
        e.printStackTrace();
    }
}

//  Get row count
public int getRowCount(){
    return model.getRowCount();
}

//  Set table data
public void setAppservName(){
    Vector data = model.getDataVector();
    Vector row = (Vector) data.elementAt(1);

    // Copy the first column
    int mColIndex = 0;-
    colData = new ArrayList(table.getRowCount()+1);

    for (int i = 0; i < table.getRowCount(); i++) {
      row = (Vector) data.elementAt(i);
      colData.add(row.get(mColIndex));
    }
}

//  Get table data
public String getAppservName(int rowNum){
    return (String) colData.get(rowNum);
}

// Set appserver info
public void setAppservInfo(String appservInfo){
    textAreaInfo.setText(appservInfo);
    textAreaInfo.setCaretPosition(0);       // Set cursor at top of text
}

// Get appserver info
public String getAppservInfo(){
    return textAreaInfo.getText();
}

/**
 * Action listeners
 */

public void addTableRowListener(ListSelectionListener listSelectionEvent) {
    table.getSelectionModel().addListSelectionListener(listSelectionEvent);
}

public void addButtonListener(ActionListener buttonEvent) {
    btnSaveInfo.addActionListener(buttonEvent);
    btnSaveInfo.setActionCommand("saveAppServInfo");
}

}
Trace output when a non zero row is selected and the refresh thread is run
** Appserver worker **
CmtModel: readAppservData
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>  Rowcount = 8

Trace output when the zero row is selected and the refresh thread is run
** Appserver worker **
CmtModel: readAppservData
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 0 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 1 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 2 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 3 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 4 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 5 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 6 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 7 <<
>>>>>>>>>  Updating the table >>>>>>>>
>>>>>>>>>  Selection list handler ! >>>>>>>>
>> Controll: selectedRow = 8 <<
java.lang.IndexOutOfBoundsException: Index: 8, Size: 8
    at java.util.ArrayList.rangeCheck(Unknown Source)
    at java.util.ArrayList.get(Unknown Source)
    at panels.ExtAppServPanel.getAppservName(ExtAppServPanel.java:219)
    at Control$SharedListSelectionHandler.valueChanged(Control.java:330)
    at javax.swing.DefaultListSelectionModel.fireValueChanged(Unknown Source)
    at javax.swing.DefaultListSelectionModel.fireValueChanged(Unknown Source)
    at javax.swing.DefaultListSelectionModel.fireValueChanged(Unknown Source)
    at javax.swing.DefaultListSelectionModel.insertIndexInterval(Unknown Source)
    at javax.swing.JTable.tableRowsInserted(Unknown Source)
    at javax.swing.JTable.tableChanged(Unknown Source)
    at javax.swing.table.AbstractTableModel.fireTableChanged(Unknown Source)
    at javax.swing.table.AbstractTableModel.fireTableRowsInserted(Unknown Source)
    at javax.swing.table.DefaultTableModel.insertRow(Unknown Source)
    at javax.swing.table.DefaultTableModel.addRow(Unknown Source)
    at javax.swing.table.DefaultTableModel.addRow(Unknown Source)
    at panels.ExtAppServPanel.setAppserverData(ExtAppServPanel.java:172)
    at Control$AppserverWorker.doInBackground(Control.java:434)
    at Control$AppserverWorker.doInBackground(Control.java:1)
    at javax.swing.SwingWorker$1.call(Unknown Source)
    at java.util.concurrent.FutureTask.run(Unknown Source)
    at javax.swing.SwingWorker.run(Unknown Source)
    at java.util.concurrent.ThreadPoolExecutor.runWorker(Unknown Source)
    at java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source)
    at java.lang.Thread.run(Unknown Source)

A: // Update GUI
theGui.extAppServPanel.setAppserverData(theModel.getAppservData());

Don't update the model or the GUI in the doInBackground() method.
All updates to the GUI should be done on the EventDispatchThread (EDT).
Instead when the data changes you need to "publish" the results so the code can be executed in the process(...) method of the SwingWorker which does execute on the EDT and therefore  the GUI will be updated on the EDT.
Read the section from the Swing tutorial on Concurrency for more information. The section on Tasks That Have Intermediate Results has an example of the publish approach.


text:
Gabaloeca retifera är en insektsart som beskrevs av Walker 1870. Gabaloeca retifera ingår i släktet Gabaloeca och familjen sköldstritar. Inga underarter finns listade i Catalogue of Life.

Källor

Sköldstritar
retifera

redpajama_set_name: RedPajamaWikipedia


Check out How to Make French Toast Restaurant Style by Huang Kitchen on Snapguide.
French toast, also known as eggy bread or gypsy toast is a dish of bread soaked in beaten egg batter, then fried and baked. As it is mostly bread, the kind of bread you use is important. Pick your favourite bread – White, Italian, French or Whole Wheat. Challah or egg bread will give incredibly creamy insides. Country- style breads are great too, and will give a sturdier, less creamy French toast. Classic French baguette sliced diagonally make a nice, chewy version. Do avoid using white sandwich bread and rustic breads with large holes and thick crust as the rustic bread can't hold the egg mixture and white bread is too soft. Also day-old or slightly stale bread is preferred for this recipe.
To make french toast, first cut the multigrain french bread into 6 thick slices.
Measure ingredients needed to fry the bread.
Place 2 eggs in a big shallow bowl.
Add the milk. Then add the ground cinnamon and salt.
Next add the brown sugar. Followed by vanilla extract.
Whisk everything together in the shallow bowl.
Dip each slice of thick french bread into the egg mixture.
Soak the bread on both sides. Make sure the bread is completely soaked.
Soak all the 6 slices of bread and drained.
Place all the soaked slices of bread on a plate.
(Optional): Set aside the soaked bread in the fridge for 10 minutes and resoaked before frying.
Preheat oven to 200 degree C (400 degree F).
Working in batches, brown the bread very slightly until golden brown. Sauté for about 3 minutes, just to kind of seal the egg batter.
Flip over and brown for another 3 minutes until it's beautiful and golden brown.
Transfer the golden brown slices of french bread to a baking sheet.
Then bake in the preheated oven for 12 to 15 minutes to finish the cooking. The bread will puff slightly and will be crispy on the outside while the inside will be incredibly moist and soft.
Keep the french toast warm in the oven over low temperature if not eaten right away.
The baking cooks the custard inside the bread and gives it an unbelievable texture. The outside on the other hand is crispy and golden. This gives a truly amazing french toast experience.
Preferably to use day-old or slightly stale bread – soak up more egg mixture without falling apart.
Cut the bread into thick slices, 1/2 to 3/4 inch thick – this helps to keep bread from becoming soggy while promoting a moist and creamy interior.
To make french toast, first cut the french bread into 6 thick slices.
Measure all ingredients needed to make the french toast.
Place the 2 eggs in a big shallow bowl.
Then add ground cinnamon and salt.
Next add the brown sugar.
Soak all the 6 slices of bread, drained and set aside.
Place all soaked slices of bread on a plate and set aside.
Working in batches, brown the bread very lightly until golden brown. Sauté for 3 minutes, just to kind of seal the egg batter.
Flip over and brown for another 3 minutes until it's beautiful and golden colour.
The baking cooks the custard inside the bread and gives it an unbelievable creamy texture. The outside on the other hand is crispy and golden. This gives a truly amazing french toast experience.
Or serve with maple syrup and garnish with some blueberries .


text:
Tim Tebow Greets Autistic Boy at On Deck Circle, Then Hits Home Run
Tim Tebow was playing baseball for the St. Lucie Mets against the Charlotte Stone Crabs at Charlotte Sports Park when he first met Seth Bosch. Tebow was on the on deck circle getting ready to step up to the plate when he heard little Seth calling his name. So he went over and shook his hand. A few minutes later, Tebow stepped up to the plate and hit a home run. The whole thing was captured by Seth's mother Ileanna on video.
The encounter started when 9-year old Seth was looking to get the attention of Tebow, one of his favorite players, from the stands. Seth has autism and hasneurofibromatosis, a genetic disorder that produces tumors on the nerve tissue. Seth started waving at Tebow, who was taking a few practice swings at the on deck circle. To everyone surprise, the star walked over and shook Seth's hand through the netting. After the encounter, Seth returned to his seat and his parents with tears of joy. What happened next was also a surprise as Tebow stepped up to the plate and hit a three-run home run for his team.
After the game, Tebow and Seth met again, where Ileanna took photos of the two together. Tim Tebow was a former professional football quarterback, where he had stints for the Denver Broncos and the New York Jets. He is currently playing for the New York Mets organization trying his hand on a professional baseball career.
"When Seth came back to his seat, he was crying," said Ileanna told the Tampa Bay Times. "And then Tim hit the homer. I started crying, too. How does that happen? I think God brought Seth and Tim together."


text:
The Croda Foundation gets a £2 million grant for health care operations
British chemicals supplier Croda has allocated a £2 million (about EUR 2.3 million) fund dedicated to improving the health of people around the world through the Croda Foundation.
Croda decided to establish a corporate foundation, which successfully achieved charity status at the end of 2021, to benefit organisations, projects and communities globally as part of its commitment to become People Positive by 2030.
So far, the Foundation has donated approximately £1m of the £2m restricted health care grant to date, supporting projects within South Asia, Africa and Brazil which are anticipated to improve over 50 million lives. The restricted grant is funding projects that aim to:
Improve vaccination delivery and infrastructure in under-served communities in five states in India, all of which have a high incidence of COVID-19 and low vaccination rates, training 22,300 personnel, providing staff for 3,700 vaccination centres and vaccinating more than 50 million people.
Provide infrastructure and training for 1,000 village-based health workers and 500 frontline staff in 20 districts in Uganda, improving the health and wellbeing of around 750,000 people.
Tackle vaccine hesitancy through education and digital inclusion, supporting 7,400 people in 10 indigenous tribes in the state of Mato Grosso, Brazil.
The Foundation has further projects in the pipeline which will be announced in due course.
Solvay Launches the First ISCC PLUS Certified Mass Balance Vanillin
LVMH announces record results in 2022 despite China


text:
What Is The Best Subscription Box For Small Dogs?
Our small pups have a ton of love to give, and what better way to return the favor than with monthly goodies? When searching for the perfect box for your furry friend, humans will want to ensure they'll receive safety-approved toys and treats that their dogs enjoy for months to come!
Here are 5 great options for monthly boxes for small dogs:
BarkBox (Because, Duh!)
Super Chewer BarkBox
The Dapper Dog
Rescue Box
Surprise My Pet
When it comes to monthly variety that the entire pack can enjoy, BarkBox is in a league of its own. From the classic Poo York City-themed box to all the holiday boxes (ho-ho-hooray!), small pups will be able to enjoy miniature flavored chews, pop open a bag of treats and fix their paws on pint-sized toys each month. When ordering, just make sure to select the 'Small & Cute' option for pups of any breed weighing 0-20 pounds.
Price: $29 for 1 month, $25 for 6 months, or $20 for 12 months.
Shipping:Free within the contiguous U.S.; $8 to Puerto Rico, Alaska, Hawaii, and Canada
The Super Chewer version of BarkBox is very similar to the one above (fun monthly themes, original products, and so on), but with the added benefit of having toys that are virtually indestructible. While most small dogs – your Yorkshire Terriers and Chihuahuas, for example – prefer something softer to play with, there are definitely those surprising petite pups who want a challenge for their teeth. THIS is the box for them.
Cost: $39 for 1 month, $33 for 3 months, $30 for 6 months, $28 for 12 months.
Shipping: Free within the contiguous U.S.; $8 to Puerto Rico, Alaska, Hawaii, and Canada.
For a dainty, classy dog, The Dapper Dog has dazzling items that are fitting for the most pampered of pooches. Like BarkBox, Dapper Dog has fun monthly themes, but the small-medium box boasts a bandana with pizzaz, too.
Price: $22-$35.
Shipping: Free to U.S.; $4.99 to Canada.
All paws on deck! For your tiny hero, here's a subscription to the rescue! Rescue Box spoils your small pup all while paying it forward to animal shelters throughout the nation! Partnered with GreaterGood.org, subscriptions provide at least 2 vaccines and 5 pounds of food to pups in need, and the love keeps growing! Your own pup 0-20 pounds can have their own personal duration of boxes with a brief description of what they'll be donating. For example, a 12 month subscription will vaccinate 24 shelter pets and provide 60 pounds of food. How's that for spreading the ruv?
Price: $24.95-$34.95.
Shipping: Free to contiguous U.S.; $4.95 to Hawaii and Alaska.
Voilà! Your wish will come true with Surprise My Pet, with boxes going as low as 0-10 pounds in your pup's weight! Subscriptions feature 5-7 items in small treats and handpicked toys, along with the possibility of your local pet-friendly organizations to receive charitable donations! While your small pup will surely be treated each month, you'll also keep the paw-sitive vibes going!
Shipping: Varies depending on location.
Is There A Dog Subscription Box With Toys Only?


text:
Thiernogryllus rufipes är en insektsart som beskrevs av Roger Roy 1969. Thiernogryllus rufipes ingår i släktet Thiernogryllus och familjen syrsor. Inga underarter finns listade i Catalogue of Life.

Källor

Syrsor
rufipes


text:
Q: Django : Call a view from a template How to call a view in my layout (template) ?
For example : I've a form on every pages of my website. I need to generate CSRF token for each pages but I don't want to put the generation code on every view.
Thank you.

A: In Django, once you reach the template, I don't believe you can call something to the effect of Zend's Action Helper.
Obviously, you could do an AJAX call to an exposed url in Django and retrieve the necessary data. In that case you can provide the csrf token to the ajax calls as follows..
$.ajaxSetup({data: {csrfmiddlewaretoken: '{{ csrf_token }}' },});

I'm not a hundred percent sure but you can implement something like Zend's Action Helper in a decorator (which could be applied to multiple views of your choice before processing the request) or in a context processor (which is applied to all views' processed request).

A: If your form is just HTML, then simply have a template containing the HTML and include that from other templates (or have it in your base template). To generate a CSRF token, you simply use {% csrf_token %} in the template, as explained at https://docs.djangoproject.com/en/dev/ref/contrib/csrf/
If you want to generate the HTML of a Django form, then you could add a context processor - explained at https://docs.djangoproject.com/en/dev/ref/templates/api/#subclassing-context-requestcontext - that generates the form and then that will be available to all templates.
def form_processor(request):
    form = Form()
    return { 'form': form.as_p() }

Template:
<form>{% csrf_token %}{{ {{ form }}</form>


"... This paper examines whether the Solow growth model is consistent with the international variation in the standard of living. It shows that an augmented Solow model that includes accumulation of human as well as physical capital provides an excellent description of the cross-country data. The paper a ..."
"... A survey of mobile and wireless technologies for ..."


text:
Home Main Story South Sudan: Striking Parliamentary Staff Suspended for Three Months
South Sudan: Striking Parliamentary Staff Suspended for Three Months
Chol Mawel
South Sudan Parliament Building
At least ten of the support staff at the national legislature who went on strike over delayed incentives recently have been suspended for three months without pay.
The suspension comes after the clerk demanded a written explanation from each of them on why they went on strike early this month.
The dissident staff were slapped with the unpaid suspension after they objected to give individual explanation, and instead chose to write the letter as a group.
The head of the workers Union at the national parliament, Youhana Wala Juma confirmed their suspension.
"Today they have issued a letter of suspension of ten out of thirteen members, it started with me as head of the union Youhana Walla Juma, Louis Christopher, Simon AKot, Sunday Joseph, Mohamed Mogga, Emmanuel Keijo, Kennedy Samuel, Kalemntina Poni and Mary Juan," Youhana said.
"We are shocked by a letter from the office of the secretary-general of the national legislature summoning some people. We already told him the explanation cannot be individual based, because we are the leadership of the workers union, and if there is anything we are the one responsible for the workers," he said.
According to the letter the group wrote, their meeting on the 6th of June was organized as a workforce briefing chaired by the R-TNLA workers union.
The union, a registered body under the national Workers Union maintains that the briefing was to share the outcome of the meeting and not a strike.
However, they explained that the Deputy Speaker for Administration and Finance at the parliament quickly called the police on suspicion that the staff were planning to go on strike, resulting in the deployment of security personnel.
Meanwhile, in a letter dated Friday last week, the august house clerk Makur Makuc Ngong wrote that the group's response was not satisfactory explanation.
Ngong then ordered for their three-month suspension without pay and also banned them from accessing the parliament premises.
"Those are the ten people who have been suspended for three months without pay and even not to enter the parliament compound, the reason they say was why we responded to the explanation as a group and supposed to be individual,' said the head of the workers union.
The suspended staff are all members of the R-TNLA Workers Union.
Eye Radio's attempts to reach the secretary general of the national parliament were not immediately successful.
However, the R-TNLA Workers Union said it will address the matter to the South Sudan Workers Union Federation.
South Sudan News
South Sudan Parliament
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South Sudan's Kiir Vows Not to Spare Coup Plotters Again


text:
require('ringo/logging').setConfig(getResource('../../log4j2.properties'));

const system = require("system");

exports.testHttpServer = require("./httpserver_test");
exports.testHttpServerBuilder = require("./builder_test");
exports.testEventSource = require("./eventsource_test");
exports.testStatic = require("./static_test");

// Test only executed outside of Github Actions:
if (system.env["SKIP_WEBSOCKET_TEST"] === undefined) {
    exports.testWebSocket = require("./websocket_test");
} else {
    console.warn("Skipping websocket_test.js");
}

if (require.main === module) {
    system.exit(require("test").run.apply(null,
        [exports].concat(system.args.slice(1))));
}

redpajama_set_name: RedPajamaGithub


FRISCHER-Leonard. Of Oyster Bay, N.Y. on August 17, 1998. Beloved husband of Ada. Devoted father of Harry and father-in-law of Debra. Cherished father of the late Barbara Frischer Brooks and father-in-law of Barry Brooks. Loving grandfather of Benjamin, Carolyn, Melissa and Casey.


The new Truck-Lite LED headlamps were initially developed for military use and offer increased durability, visibility and safety to the heavy-duty truck platform. Manufactured in the USA, Truck-Lite's forward-lighting technology has been proven on vehicles serving in Iraq and Afghanistan since 2007.
For trucks other than the Freightliner Cascadia, easy-to-install aftermarket retrofit options include traditional 7" round and 5"x7" alternatives.
Truck-Lite's LED forward- lighting headlamps offer improved driver visibility through its more natural light spectrum, leading to improved object recognition at night and increased daytime visibility. Truck-Lite's LED beam pattern also reduces eye-strain for drivers and reduces harsh headlight glare to oncoming traffic. An additional benefit is a significant reduction in amperage draw on the vehicles electrical system, freeing up power for other uses and resulting in a 50-times longer lighting system life than conventional halogen technology.