The solution structure of the ADAR2 dsRBM-RNA complex reveals a sequence-specific readout of the minor groove.

Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.

Educators' Perspectives on Animal Welfare and Ethics in the Australian and New Zealand Veterinary Curricula.

The current study was designed to explore the importance that veterinary science educators in Australian and New Zealand universities assign to animal welfare and ethics (AWE) topics as Day One/Initial Competences for new graduates. An online questionnaire was deployed in parallel with an equivalent study of veterinary science students at these educators' schools. Responses were received from 142 educators (51% females n=72 and 49% males n=70), representing an overall participation rate of 25%. Questions were clustered according to seven areas of veterinary employment: general practice, production animals, companion animals, wild animals, aquatic animals, animals kept for scientific purposes, and animals used in sport and recreation. The most highly rated topics for each of these clusters were: professional ethics in general practice, euthanasia in companion animals, strategies to address painful husbandry procedures in production animals, veterinarians' duties to wild animals in animals in the wild, aquatic animal health and welfare issues in aquatic animals; competence in the 3Rs (replacement, refinement and reduction) in animals kept for scientific purposes, and responsibilities of ownership in sport and recreation. Female educators rated many of the topics as significantly more important than did their male counterparts. Educators teaching one or more ethics-related subjects were less likely to rate neutering and euthanasia as important as those not teaching these subjects. The educators' focus on practical issues clashes with a perceived need for veterinarians to actively embrace animal ethics. Overall, the perspectives of these educators should be carefully considered as they are likely to influence student attitudes.

Regulatory and T Effector Cells Have Overlapping Low to High Ranges in TCR Affinities for Self during Demyelinating Disease.

Having regulatory T cells (Tregs) with the same Ag specificity as the responding conventional T cells is thought to be important in maintaining peripheral tolerance. It has been demonstrated that during experimental autoimmune encephalomyelitis there are myelin oligodendrocyte glycoprotein (MOG)--specific Tregs that infiltrate into the CNS. However, the affinity of naturally occurring polyclonal Tregs for any self-antigen, let alone MOG, has not been analyzed in the periphery or at the site of autoimmune disease. Utilizing the highly sensitive micropipette adhesion frequency assay, which allows one to determine on a single-cell basis the affinity and frequency of polyclonal Ag-specific T cells directly ex vivo, we demonstrate that at peak disease MOG-specific Tregs were progressively enriched in the draining cervical lymph nodes and CNS as compared with spleen. These frequencies were greater than the frequencies measured by tetramer analysis, indicative of the large fraction of lower affinity T cells that comprise the MOG-specific conventional T cell (Tconv) and Treg response. Of interest, the self-reactive CD4(+) Tconvs and Tregs displayed overlapping affinities for MOG in the periphery, yet in the CNS, the site of neuroinflammation, Tconvs skew toward higher affinities. Most of the MOG-specific Tregs in the CNS possessed the methylation signature associated with thymic-derived Tregs. These findings indicate that thymic-derived Treg affinity range matches that of their Tconvs in the periphery and suggest a change in TCR affinity as a potential mechanism for autoimmune progression and escape from immune regulation.

The Importance of Animal Welfare Science and Ethics to Veterinary Students in Australia and New Zealand.

The study of animal welfare and ethics (AWE) as part of veterinary education is important due to increasing community concerns and expectations about this topic, global pressures regarding food security, and the requirements of veterinary accreditation, especially with respect to Day One Competences. To address several key questions regarding the attitudes to AWE of veterinary students in Australia and New Zealand (NZ), the authors surveyed the 2014 cohort of these students. The survey aimed (1) to reveal what AWE topics veterinary students in Australia and NZ consider important as Day One Competences, and (2) to ascertain how these priorities align with existing research on how concern for AWE relates to gender and stage of study. Students identified triage and professional ethics as the most important Day One Competences in AWE. Students ranked an understanding of triage as increasingly important as they progressed through their program. Professional ethics was rated more important by early and mid-stage students than by senior students. Understanding the development of animal welfare science and perspectives on animal welfare were rated as being of little importance to veterinary graduates as Day One Competences, and an understanding of "why animal welfare matters" declined as the students progressed through the program. Combined, these findings suggest that veterinary students consider it more important to have the necessary practical skills and knowledge to function as a veterinarian on their first day in practice.

Career Preferences and Opinions on Animal Welfare and Ethics: A Survey of Veterinary Students in Australia and New Zealand.

Historically, the veterinary profession has understood animal welfare primarily in terms of animal health and productivity, with less recognition of animals' feelings and mental state. Veterinary students' career preferences and attitudes to animal welfare have been the focus of several international studies. As part of a survey in Australia and New Zealand, this study reports on whether veterinary students prioritize animal welfare topics or professional conduct on the first day of practice and examines links between students' career preferences and their institution, gender, and year of study. The questionnaire was designed to explore the importance that students assign to topics in animal welfare and ethics. Of the 3,320 students invited to participate in the online survey, a total of 851 students participated, representing a response rate of 25.5%. Students' preferences increased for companion-animal practice and decreased for production-animal practice as they progressed through their studies. Females ranked the importance of animal welfare topics higher than males, but the perceived importance declined for both genders in their senior years. In line with previous studies, this report highlighted two concerns: (1) the importance assigned to animal welfare declined as students progressed through their studies, and (2) males placed less importance overall on animal welfare than females. Given that veterinarians have a strong social influence on animal issues, there is an opportunity, through enhanced education in animal welfare, to improve student concern for animal welfare and in turn improve animal care and policy making by future veterinarians.

Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions.

Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neurotropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo.

Editing of neurotransmitter receptor and ion channel RNAs in the nervous system.

The central dogma of molecular biology defines the major route for the transfer of genetic information from genomic DNA to messenger RNA to three-dimensional proteins that affect structure and function. Like alternative splicing, the post-transcriptional conversion of adenosine to inosine (A-to-I) by RNA editing can dramatically expand the diversity of the transcriptome to generate multiple, functionally distinct protein isoforms from a single genomic locus. While RNA editing has been identified in virtually all tissues, such post-transcriptional modifications have been best characterized in RNAs encoding both ligand- and voltage-gated ion channels and neurotransmitter receptors. These RNA processing events have been shown to play an important role in the function of the encoded protein products and, in several cases, have been shown to be critical for the normal development and function of the nervous system.

Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi syndrome.

RNA transcripts encoding the 2C-subtype of serotonin (5HT(2C)) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT(2C) editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT(2C) receptor expression and 5HT(2C)-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT(2C) gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT(2C) expression, but also demonstrate the importance of normal patterns of 5HT(2C) RNA editing in vivo.

Immunoprofiling toll-like receptor ligands: Comparison of immunostimulatory and proinflammatory profiles in ex vivo human blood models.

There is a pressing need for the development of novel, safe and effective adjuvants. The recent discovery and characterization of pathogen-associated molecular pattern (PAMP)-recognizing elements such as the Toll-like, NOD-like and RIG-like receptors, has brought into sharp focus the role of PAMPs in bridging the innate and adaptive immune responses, and a detailed understanding of the immunostimulatory vis-à-vis proinflammatory activities could lead to the development of effective adjuvants, monophosphoryl lipid A being an excellent example. We describe in this paper a series of hierarchical assays that were employed to characterize TLR agonists in vitro including primary TLR-reporter assays, secondary indices of immune activation, and tertiary screens characterizing transcriptomal activation patterns to identify optimal immunostimulatory chemotypes. The evaluation of representative members of known human TLR agonists demonstrate that TLR2, -4, -5 and -7 agonists were immunostimulatory. TLR7 agonists were extremely immunostimulatory, stimulating nearly all subsets of lymphocytes without inducing proinflammatory cytokine responses. The TLR5 agonist, flagellin, while immunostimulatory, was also highly proinflammatory. These results suggest that TLR agonists other than lipid A-like chemotypes could be developed into potential adjuvants, and that this series of hierarchical assays could be adapted to rapidly identify in large libraries, compounds with adjuvantic potential that lack proinflammatory responses.

A Hybrid Insulin Epitope Maintains High 2D Affinity for Diabetogenic T Cells in the Periphery.

ß-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29 -42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.

Review of the Online One Welfare Portal: Shared Curriculum Resources for Veterinary Undergraduate Learning and Teaching in Animal Welfare and Ethics.

This article introduces the online One Welfare learning and teaching portal (OWP) and describes its development, use, importance and relevance to animal welfare and ethics (AWE) stakeholders. As animal welfare issues increase in importance, veterinarians must be trained to lead the science that underpins AWE discourses. The OWP is a collection of resources designed to engage and challenge veterinary science students as they become advocates for animals. It was developed collaboratively by all eight veterinary schools in Australia and New Zealand, and funded by the Australian Government Office for Learning and Teaching. Surveys to investigate the attitudes of students and educators to AWE issues in six context-specific themes based on the Australian Animal Welfare Strategy (AAWS) (companion animals; animals used in research and teaching; livestock/production animals; animals used for sport, recreation or display; animals in the wild and aquatic animals) were administered through all participating schools. Students assigned more importance to Day One competence in knowledge of welfare concepts than did educators for the following groups: production animals, companion animals, animals in the wild, aquatic animals, animals used in research and teaching, and animals used for sport, recreation or display (all p < 0.01). Agreement between educators and students was closer regarding the importance of Day One competence for euthanasia for all six context-specific themes (p < 0.01 - 0.03). Students assigned more importance than educators to social, economic and cultural drivers of welfare outcomes in production animals (p < 0.01); slaughter and preslaughter inspections in production animals (p < 0.01); animal abuse and hoarding in companion animals (p < 0.01); shelter medicine in companion animals (p < 0.01); disaster preparedness in wildlife animals (p < 0.01); pain and distress caused by fishing in aquatic animals (p < 0.01); conscientious objection related to animals held for research and teaching (p < 0.01); behaviour, selection and training of animals used for sport, recreation and display (p = 0.046) and educating the public around sporting animal welfare (p < 0.01). Agreement between educators and students was closer for strategies to address painful husbandry procedures in production animals (p = 0.03); behaviour and training of companion animals (p = 0.03); veterinarians' duties to wild animals in wildlife (p = 0.02); the 3Rs in animals held for research and teaching (p = 0.03) and ownership responsibility in sporting animals (p = 0.01). This report discusses the reasons for differences among students and educators as they approach these issues. The portal is expected to gather more content as veterinary schools in other countries use its resources and users submit scenarios and discussion topics that reflect local needs.

Potential adjuvantic properties of innate immune stimuli.

Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns and serve as primary sensors of the innate immune system. Ten members of the TLR family have so far been identified in the human genome. The ligands for these receptors are structurally highly conserved microbial molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double-stranded RNA (TLR3), CpG motif-containing DNA (TLR9) and profilin present on uropathogenic bacteria (TLR 11). Complementing the TLRs are the nucleotide-binding domain (NOD), leucine rich repeat containing family (or Nod-like Receptors, NLRs), which detect muramylpeptides released from bacterial peptidoglycan (PGN) in the intracytoplasmic compartment, as well as the retinoic-acid-inducible protein 1 (RIG-I-like receptors; RLRs) which sense single-stranded RNA of viral origin. The activation of PRRs by their cognate ligands leads to production of inflammatory cytokines, upregulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer cells, in addition to priming and amplifying antigen-specific T-, and B-cell effector functions. Thus, these stimuli serve to link innate and adaptive immunity and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses. This review summarizes what is currently known about the immunopotentiatory and adjuvantic activities of innate immune stimuli.

A Pilot Welfare Assessment of Working Ponies on Gili Trawangan, Indonesia.

Many working equids in developing countries experience poor health and welfare due to environmental and management factors. Collecting baseline data on these populations is essential to inform education projects to improve equid health and welfare. Gili Trawangan is an island in Indonesia that has no motor vehicles and a working pony population of approximately 200. This pilot study sought to determine baseline data on their health, welfare, and husbandry to inform future health and welfare strategies. A cross-sectional study was performed in May 2017 consisting of a pony cart driver questionnaire (n = 33) and a basic pony health examination (n = 38). The body condition scores of ponies were 3/5 (60.5%), 2/5 (31.6%), and 1/5 (7.9%), while 38% and 92% of ponies had lameness and foot pathology, respectively, and 31 ponies (86.1%) had at least one wound. Thirty percent of cart drivers stated they would work their ponies despite evidence of injury or illness. Limited education, poor access to veterinary services, and remoteness pose barriers to improving pony health and welfare. Our results indicate a need for, and can help inform, targeted education programmes to improve the lives of working ponies and protect livelihoods and tourism on Gili Trawangan.

Comparison of the immunostimulatory and proinflammatory activities of candidate Gram-positive endotoxins, lipoteichoic acid, peptidoglycan, and lipopeptides, in murine and human cells.

The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and its identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-kappaB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-kappaB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably lower than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS.

Importance of Welfare and Ethics Competence Regarding Animals Kept for Scientific Purposes to Veterinary Students in Australia and New Zealand.

Veterinarians are in a strong position of social influence on animal-related issues. Hence, veterinary schools have an opportunity to raise animal health and welfare standards by improving veterinary students' animal welfare and ethics (AWE) education, including that related to animals used for scientific purposes. A survey of 851 students in the early, mid, and senior stages of their courses at all eight veterinary schools across Australia and New Zealand was undertaken on their first day of practice (or Day One Competences) to explore how veterinary students viewed the importance of their competence in the management of welfare and ethical decision-making relating to animals kept for scientific purposes. From highest to lowest, the rankings they assigned were: Animal Ethics Committee (AEC) Procedures or Requirements; 3Rs (Replacement, Refinement and Reduction); Humane Endpoints; Euthanasia; "What Is a Research Animal?"; and Conscientious Objections. Female students rated Conscientious Objections, Humane Endpoints, and Euthanasia significantly higher than male students did across the three stages of study. The score patterns for these three variates showed a trend for the male students to be more likely to score these topics as extremely important as they advanced through the course, but female students' scores tended to decline slightly or stay relatively stable. No gender differences emerged for the three variates: 3Rs (Replacement, Refinement and Reduction); AEC Procedures or Requirements; and "What Is a Research Animal?". This study demonstrates that understandings of the regulatory and normative frameworks are considered most important in animal welfare and ethics competence in veterinary students. To the authors' knowledge, this is the first study to investigate what importance veterinary students place on their competence regarding animals kept for scientific purposes.

Ranking of Production Animal Welfare and Ethics Issues in Australia and New Zealand by Veterinary Students.

The importance of animal welfare and ethics (AWE) within the veterinary education should reflect community concerns and expectations about AWE, and the professional demands of veterinary accreditation on the first day of practice (or 'Day One' competences). Currently, much interest and debate surrounds the treatment of production animals, particularly around live export. To explore the attitudes to AWE of veterinary students in Australia and New Zealand, a survey was undertaken to (i) understand what students consider important AWE topics for initial production animal competence; and (ii) ascertain how these priorities correlated with gender, area of intended practice and stage-of-study. The results from 575 veterinary students showed that all students ranked strategies to address painful husbandry procedures as the most important issues on their first day in production animal practice. Additionally, it was found that the importance students assigned to an understanding of human⁻animal interactions declined as they progressed through the veterinary course. In contrast, the importance of an understanding of euthanasia issues for production animals increased for male students as they progressed through the course, and remained consistently high in females. Females also gave higher ranking to the importance of understanding production animal stress associated with transport, and ranked strategies to address painful husbandry procedures more important than did males. These findings should help the development of AWE teaching resources that address students' attitudes and competence and that can be delivered when students are most receptive.

Whip Rule Breaches in a Major Australian Racing Jurisdiction: Welfare and Regulatory Implications.

Whip use in horseracing is increasingly being questioned on ethical, animal welfare, social sustainability, and legal grounds. Despite this, there is weak evidence for whip use and its regulation by Stewards in Australia. To help address this, we characterised whip rule breaches recorded by Stewards using Stewards Reports and Race Diaries from 2013 and 2016 in New SouthWales (NSW) and the Australian Capital Territory (ACT). There were more recorded breaches at Metropolitan (M) than Country (C) or Provincial (P) locations, and by riders of horses that finished first, second, or third than by riders of horses that finished in other positions. The most commonly recorded breaches were forehand whip use on more than five occasions before the 100-metre (m) mark (44%), and whip use that raises the jockey's arm above shoulder height (24%). It is recommended that racing compliance data be analysed annually to inform the evidence-base for policy, education, and regulatory change, and ensure the welfare of racehorses and racing integrity.

The First Shared Online Curriculum Resources for Veterinary Undergraduate Learning and Teaching in Animal Welfare and Ethics in Australia and New Zealand.

The need for undergraduate teaching of Animal Welfare and Ethics (AWE) in Australian and New Zealand veterinary courses reflects increasing community concerns and expectations about AWE; global pressures regarding food security and sustainability; the demands of veterinary accreditation; and fears that, unless students encounter AWE as part of their formal education, as veterinarians they will be relatively unaware of the discipline of animal welfare science. To address this need we are developing online resources to ensure Australian and New Zealand veterinary graduates have the knowledge, and the research, communication and critical reasoning skills, to fulfill the AWE role demanded of them by contemporary society. To prioritize development of these resources we assembled leaders in the field of AWE education from the eight veterinary schools in Australia and New Zealand and used modified deliberative polling. This paper describes the role of the poll in developing the first shared online curriculum resource for veterinary undergraduate learning and teaching in AWE in Australia and New Zealand. The learning and teaching strategies that ranked highest in the exercise were: scenario-based learning; a quality of animal life assessment tool; the so-called 'Human Continuum' discussion platform; and a negotiated curriculum.

Insights into T cell recognition of antigen: significance of two-dimensional kinetic parameters.

The T cell receptor (TCR) interacts with peptide-major histocompatibility complex (pMHC) to enable T cell development and trigger adaptive immune responses. For this reason, TCR:pMHC interactions have been intensely studied for over two decades. However, the details of how various binding parameters impact T cell activation remain elusive. Most measurements were made using recombinant proteins by surface plasmon resonance, a three-dimensional (3D) technique in which fluid-phase receptors and ligands are removed from their cellular environment. This approach found TCR:pMHC interactions with relatively low affinities and slow off-rates for agonist peptides. Newer generation techniques have analyzed TCR:pMHC interactions in two dimensions (2D), with both proteins anchored in apposing plasma membranes. These approaches reveal in situ TCR:pMHC interaction kinetics that are of high affinity and exhibit rapid on- and off-rates upon interaction with agonist ligands. Importantly, 2D binding parameters correlate better with T cell functional responses to a spectrum of ligands than 3D measures.

Low 2-dimensional CD4 T cell receptor affinity for myelin sets in motion delayed response kinetics.

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling--no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity--as well as delayed CD69 and CD25 expression (12-24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.