Several common genetic variations have been associated with type 2 diabetes, but the exact disease mechanisms are still poorly elucidated. Using congenic strains from the diabetic Goto-Kakizaki rat, we identified a 1.4-megabase genomic locus that was linked to impaired insulin granule docking at the plasma membrane and reduced beta cell exocytosis. In this locus, Adra2a, encoding the alpha2A-adrenergic receptor [alpha(2A)AR], was significantly overexpressed. Alpha(2A)AR mediates adrenergic suppression of insulin secretion. Pharmacological receptor antagonism, silencing of receptor expression, or blockade of downstream effectors rescued insulin secretion in congenic islets. Furthermore, we identified a single-nucleotide polymorphism in the human ADRA2A gene for which risk allele carriers exhibited overexpression of alpha(2A)AR, reduced insulin secretion, and increased type 2 diabetes risk. Human pancreatic islets from risk allele carriers exhibited reduced granule docking and secreted less insulin in response to glucose; both effects were counteracted by pharmacological alpha(2A)AR antagonists.

Pristane-induced arthritis (PIA) in rats, a model for rheumatoid arthritis (RA), is a T cell-dependent disease. However, pristane itself is a lipid and unable to form a stable complex with a MHC class II molecule. Therefore, the specificity and function of the T cells in PIA are as unclear as in rheumatoid arthritis. In this study, we show that activated CD4+ αβT cells, which target peripheral joints, transfer PIA. The pristane-primed T cells are of oligo or polyclonal origin as determined by their arthritogenicity after stimulation with several mitogenic anti-TCRVβ and anti-TCRVα mAbs. Arthritogenic cells secreted IFN-γ and TNF-α (but not IL-4) when stimulated with Con A in vitro, and pretreatments of recipient rats with either anti-IFN-γ or a recombinant TNF-α receptor before transfer ameliorated arthritis development. Most importantly, we show that these T cells are MHC class II restricted, because treatment with Abs against either DQ or DR molecules ameliorates arthritis development. The MHC class II restriction was confirmed by transferring donor T cells to irradiated recipients that were syngenic, semiallogenic, or allogenic to MHC class II molecules, in which only syngenic and semiallogenic recipients developed arthritis. These data suggest that the in vivo administration of a non-antigenic adjuvant, like pristane, activates CD4+ αβT cells that are MHC class II restricted and arthritogenic.

BackgroundTo facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data.MethodsWe optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies.ResultsOut of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment.ConclusionsPIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.

The laboratory rat (Rattus norvegicus) is a key tool for the study of medicine and pharmacology for human health. A large database of phenotypes for integrated fields such as cardiovascular, neuroscience, and exercise physiology exists in the literature. However, the molecular characterization of the genetic loci that give rise to variation in these traits has proven to be difficult. Here we show how one obstacle to progress, the fine-mapping of quantitative trait loci (QTL), can be overcome by using an outbred population of rats. By use of a genetically heterogeneous stock of rats, we map a locus contributing to variation in a fear-related measure (two-way active avoidance in the shuttle box) to a region on chromosome 5 containing nine genes. By establishing a protocol measuring multiple phenotypes including immunology, neuroinflammation, and hematology, as well as cardiovascular, metabolic, and behavioral traits, we establish the rat HS as a new resource for the fine-mapping of QTLs contributing to variation in complex traits of biomedical relevance.The rat has for long been a favored organism for physiological and behavioral analyses and is increasingly attracting the attention of geneticists (Jacob and Kwitek 2002). Over the last century, a wealth of disease models have been developed, which compared with the mouse have proved easier to analyze at an organ and cellular level because of the rat's larger size. Rat models of cardiovascular disease, inflammatory diseases, and susceptibility to cancer and toxic substances have been crucial in understanding the biology of common human disorders. The rat has also been a focus of classical neuroanatomical studies and electrophysiological slice studies; rat experiments have been critical for understanding many neurobiological processes, including learning and memory, and for providing models for the neuropsychology of human behavioral disorders (Weiss and Feldon 2001).

A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) is driven by autoreactive T cells but no autoantigen has been identified to date. We therefore analyzed B and T cell responses to autoantigens potentially involved in the pathogenesis of RA, including IgG, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (RA33). IgG and IgM autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed DA.1F rats already 1 wk before disease onset, reached maximum levels during the acute phase, and correlated with arthritis severity. Apart from rheumatoid factor, autoantibodies to other Ags were not observed. CD4+ lymph node cells isolated 10 days after pristane injection produced IFN-γ but not IL-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate Ags elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated lymph node cells of naive animals to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in the joints of rats injected with pristane. Overexpression coincided with the appearance of anti-RA33 Abs and preceded the onset of clinical symptoms of PIA by several days. Taken together, these data suggest hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this Ag might play a pivotal role in the pathogenesis of PIA and possibly also human RA.

Objective. To investigate type II collagen (CII) as a joint-specific target of the anti-citrullinated protein antibody (ACPA) response in rheumatoid arthritis (RA).Methods. Potential citrullinated neoepitopes were identified by high-resolution tandem mass spectrometry (MS/MS) of in vitro peptidylarginine deiminase 2 (PAD-2)-treated CII, and the relationship between citrullination and CII conformation was investigated by circular dichroism and conformation-dependent antibodies. Based on the MS analyses, synthetic peptides were designed and analyzed for serum IgG reactivity in the Epidemiological Investigation of RA (EIRA) casecontrol cohort of 1,949 RA patients and 278 healthy controls. Peptide-specific antibodies were purified from RA patient serum and used to stain RA cartilage specimens.Results. We described the conformationdependent citrullination pattern of CII after PAD-2 treatment at room temperature and 37°C and showed that CII could be citrullinated in its native triple-helical conformation. Screening of Arg and Cit pairs of synthetic peptides revealed new citrullinated B cell epitopes on CII. Antibodies directed to 2 proximal epitopes close to the C-terminus of the CII triple helix were recognized by autoantibodies in 21% and 17% of RA patients, respectively. Affinity-purified antibodies from RA sera directed to these 2 epitopes, but not antibodies directed to citrullinated ␣-enolase peptide 1, bound to RA cartilage.Conclusion. These findings suggest that cartilagedirected anticitrulline immunity contributes to the induction of joint inflammation in RA.

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

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