Role of DLA-DRB1 amino acids outside the shared epitope in dachshund susceptibility to immune-mediated polyarthritis.

Canine immune-mediated polyarthritis (IMPA) is an idiopathic disorder encompassing both erosive and non-erosive forms of rheumatoid arthritis (RA), with a clinical picture similar to human RA. Resemblance in major histocompatibility complex (MHC)-associated risk between the two was first noted within the specific amino acid motif known as the shared epitope (SE) on human leukocyte antigen DRB1. Following further identification of amino acids conferring risk for human RA outside the SE, this study was designed to examine amino acids both within and outside the classic SE in dachshunds, a breed with reported susceptibility to IMPA in Japan. Genome-wide association studies have linked positions 11, 13 and 71 with strong risk for human RA and important roles in antigen presentation to T cells. Sequence based genotyping of 16 case and 64 control dachshunds revealed strong associations comparable to human RA between IMPA risk and valine at position 11 (Val-11), phenylalanine at 13 (Phe-13), and arginine at 71 (Arg-71) on the dog leukocyte antigen (DLA)-DRB1 molecule (OR 2.89, 95%CI 1.3-6.4, p = 0.009), while association with the classic SE was significant only regarding homozygote frequency of the QRRAA haplotype-also carrying Val 11 and Phe 13 outside the SE (p = 0.04). Moreover, limited range in possible combinations of amino acids at positions 11, 13 and 71 starting with Val-11 among all DLA-DRB1 alleles registered with the GenBank and IPD-MHC canine databases, suggested potential of further single-breed analyses in dachshunds to clarify the disorder in terms of diagnosis, treatment, and epigenetic control, while clinical and immunopathogenetic similarities between human and dachshund RA also suggested the possibility of gaining insight into RA per se through study of canine IMPA as a spontaneous model of human RA.

Long-term treatment of allogeneic adipose-derived stem cells in a dog with rheumatoid arthritis.

BACKGROUND: Although there are growing demands for stem cell-based therapy for companion animals in various diseases, a few clinical trials have been reported. Moreover, most of them are the results from only one or a few times of stem cell injection. OBJECTIVES: The aim of this study is to describe a long-term treatment with allogeneic adipose-derived stem cells (ASCs) in a dog with rheumatoid arthritis (RA), which is a rare canine disease. METHODS: The dog with RA received intravascular injection of allogeneic ASCs derived from two healthy donors once a month for 11 months. To assess therapeutic effects of ASCs, orthopedic examination and clinical evaluation was performed. Cytokines of tumor necrosis factor-α and interleukin-6 in the plasma were measured using ELISA analysis. RESULTS: Despite this repeated and long-term administration of allogeneic ASCs, there were no side effects such as immunorejection responses or cell toxicity. The orthopedic examination score for the dog decreased after ASCs treatment, and the clinical condition of the dog and owner's satisfaction were very good. CONCLUSIONS: Although ASCs has been suggested as one of the options for RA treatment because of its anti-inflammatory and immunosuppressive functions, it has never been used to treat RA in dogs. The present report describes a case of canine RA treated with allogeneic ASCs for long-term in which the dog showed clinical improvement without adverse effects.

Effect of nabumetone on humoral immune responses in mice / Efeito da nabumetona nas respostas imunes humorais em camundongos

Nabumetone is used to reduce the pain and inflammation in rheumatoid arthritis. In the current study, immunomodulatory effect of Nabumetone is investigated in mice. The control group was administered normal saline orally as placebo. Nabumetone was administered orally via gavage in two treatment groups at 14mg/kg.b.w. doses and 28mg/kgb.w., respectively. Haemagglutination (HA) assay, Jerne hemolytic plaque and mice lethality assays were applied. In HA assay, the titer was significantly decreased in Nabumetone treatment groups (P< 0.001). In Jerne hemolytic plaque formation assay, there was a significant reduction (P< 0.001) in number of plaques in Nabumetone treated groups when compared with control. In mice lethality assay, there was a significant difference in mortality ratio of mice in control and Nabumetone treated groups (P< 0.001). Therefore, it is concluded that Nabumetone suppresses the humoral immune response in mice.(AU)

A nabumetona é usada na redução da dor e inflamação da artrite reumática. No presente estudo, o efeito imunomodulador é investigado em camundongos. O grupo de controle recebeu solução salina via oral como placebo. Nabumetona foi administrada oralmente via gavagem em dois grupos de tratamentos com doses de 14mg/kg.b.w. e 28mg/kgb.w., respectivamente. Foram realizados ensaios de hemaglutinação (HA), placa hemolítica de Jerne e letalidade dos camundongos. No ensaio HA, o grau foi significativamente menor nos grupos de tratamento com nabumetoma (P< 0.001). No ensaio de formação de placa hemolítica de Jerne houve redução significativa (P< 0.001) no número de placas em grupos tratados com nabumetoma comparado ao controle. No ensaio de letalidade dos camundongos houve diferença significativa no grau de mortalidade de camundongos no grupo de controle e grupos tratados com nabumetoma (P< 0.001). Portanto, conclui-se que a Nabumetoma suprime a resposta imune humoral em camundongos.(AU)

Synovial fluid matrix metalloproteinase-2 and -9 activities in dogs suffering from joint disorders.

The activity of matrix metalloproteinase (MMP)-2 and MMP-9 in synovial fluids (SF) sampled from dogs with joint disorders was investigated by gelatin zymography and densitometry. Pro-MMP-2 showed similar activity levels in dogs with idiopathic polyarthritis (IPA; n=17) or canine rheumatoid arthritis (cRA; n=4), and healthy controls (n=10). However, dogs with cranial cruciate ligament rupture (CCLR; n=5) presented significantly higher pro-MMP-2 activity than IPA and healthy dogs. Meanwhile, dogs with IPA exhibited significantly higher activity of pro- and active MMP-9 than other groups. Activity levels in pro- and active MMP-9 in cRA and CCLR dogs were not significantly different from those in healthy controls. Different patterns of MMP-2 and MMP-9 activity may reflect the differences in the underlying pathological processes.

Comparison of the efficacy of prednisone and cyclosporine for treatment of dogs with primary immune-mediated polyarthritis.

OBJECTIVE: To compare efficacy between cyclosporine and prednisone for treatment of primary immune-mediated polyarthritis (IMPA) in dogs. DESIGN: Randomized controlled clinical trial. ANIMALS: 20 client-owned dogs with primary IMPA. PROCEDURES: Dogs were randomly assigned to receive prednisone (starting at 1 mg/kg [0.45 mg/lb], PO, q 12 h; n = 10) or cyclosporine (5 mg/kg [2.3 mg/lb], PO, q 12 h; 10) for 90 days. Cyclosporine-treated dogs also received carprofen, tramadol, or both for the first 7 days for analgesia. Data collection, physical examination, and cytologic analysis of synovial fluid samples were performed on days 0, 14, 45, and 90. Trough whole blood cyclosporine concentrations were determined on days 7 to 17 for cyclosporine-treated dogs. Treatment failure was defined as lack of clinical improvement by day 14, lack of cytologic improvement by day 45, or need to change treatment because of adverse effects. RESULTS: Treatment was successful for 7 prednisone-treated dogs and 7 cyclosporine-treated dogs. Absence of synovial fluid cytologic abnormalities on day 45 was identified for 5 prednisone-treated dogs and 8 cyclosporine-treated dogs. Prednisone-treated dogs were more likely to develop polyuria, polydipsia, and polyphagia than were cyclosporine-treated dogs. Opportunistic infections (ie, demodicosis or Erysipelothrix bacteremia) were identified in 2 cyclosporine-treated dogs and 0 prednisone-treated dogs, and diarrhea developed in 1 cyclosporine-treated dog, requiring treatment discontinuation. CONCLUSIONS AND CLINICAL RELEVANCE: Although the number of dogs evaluated was small, limiting generalizability, results of this study suggested that cyclosporine offers promise as a suitable alternative to prednisone for treatment of IMPA in dogs.

Mouse Models of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder characterized by synovitis that leads to cartilage and bone erosion by invading fibrovascular tissue. Mouse models of RA recapitulate many features of the human disease. Despite the availability of medicines that are highly effective in many patient populations, autoimmune diseases (including RA) remain an area of active biomedical research, and consequently mouse models of RA are still extensively used for mechanistic studies and validation of therapeutic targets. This review aims to integrate morphologic features with model biology and cover the key characteristics of the most commonly used induced and spontaneous mouse models of RA. Induced models emphasized in this review include collagen-induced arthritis and antibody-induced arthritis. Collagen-induced arthritis is an example of an active immunization strategy, whereas antibody- induced arthritis models, such as collagen antibody-induced arthritis and K/BxN antibody transfer arthritis, represent examples of passive immunization strategies. The coverage of spontaneous models in this review is focused on the TNFΔ (ARE) mouse, in which arthritis results from overexpression of TNF-α, a master proinflammatory cytokine that drives disease in many patients.

Invasion, stress, and spinal arthritis in cane toads.

The impact of invasive species on biodiversity has attracted considerable study, but impacts of the invasion process on the invaders themselves remain less clear. Invading species encounter conditions different from those in their ancestral habitats and are subject to intense selection for rapid dispersal. The end result may be significant stress on individual organisms, with consequent health problems. Our studies on invasive cane toads in Australia reveal severe spinal arthritis in approximately 10% of large adult toads, associated with the same factors (large body size, frequent movement, and relatively long legs) that have enabled toads to invade so rapidly across the Australian tropics.

Veterinary autoimmunity: autoimmune diseases in domestic animals.

The first spontaneous animal model of autoimmunity was the New Zealand black mouse, discovered in 1959. Interestingly, although several models of induced autoimmunity were demonstrated in a variety of rodents, the recognition of autoimmune disease in dogs came somewhat later. Dog breeding and selection of traits within certain dog breeds have become an important enterprise with intensive husbandry and selection criteria being applied to each breed standard. This has resulted in breeding for specific phenotypic characteristics. This selection has inadvertently led to the propagation of a number of autoimmune diseases in dogs. For example, systemic lupus erythematosus (SLE), autoimmune hemolytic anemia and thrombocytopenia, autoimmune myasthenia gravis, and diabetes mellitus are now fairly common. In the final analysis, the appearance of autoimmunity in dogs reflects their breeding selection and illustrates the importance of genetics in the development of autoimmune disease.

Disease modifying treatment for feline rheumatoid arthritis.

Feline erosive polyarthritis includes the more common periosteal proliferative polyarthritis (PPP) and the rarely seen rheumatoid arthritis (RA) (11). During the past three years, 12 patients with definite feline rheumatoid arthritis, which did not respond well to conventional therapy, were treated with 7.5 mg of Methotrexate and 70 mg Leflunomide, given weekly by the oral route. The average age of the cats was 5.9 years (range 2.5 to 10 years). Siamese cats were over represented. Seven of the 12 (58%) cats showed a marked improvement, usually within four weeks. Once maximum improvement was obtained the dosage was decreased. Serious toxicity was not noted and carcinogenetic effect was not seen during the course of this study.

Synovial MMP-3 and TIMP-1 levels and their correlation with cytokine expression in canine rheumatoid arthritis.

The purpose of this study was to investigate whether synovial fluid levels of matrix metalloprotease-3 (MMP-3) and tissue inhibitor of metalloprotease-1 (TIMP-1) are specifically elevated in canine rheumatoid arthritis (CRA) compared to osteoarthritic joint disorders and if these markers are correlated with a specific pattern of cytokine mRNA expression. Synovial fluid samples of 17 dogs with CRA were analysed for MMP-3 and TIMP-1 by two canine sandwich ELISA (enzyme-linked immunosorbent assay) systems. The synovial mRNA content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma) was determined by RT-PCR (reverse transcription-polymerase chain reaction). Dogs with osteoarthritis (n = 50) caused by anterior cruciate ligament rupture (ACLR) were used as controls. A significant rise of MMP-3 was found in the synovial fluid of joints with CRA that could not be balanced by sufficient amounts of TIMP-1. The 30-fold surplus of MMP-3 over TIMP-1 was strongly correlated with the synovial mRNA content of IL-1, IL-12 and TGF-beta. Our results point to the potential use of the synovial levels of MMP-3 as a marker for RA in dogs.

Dog MHC alleles containing the human RA shared epitope confer susceptibility to canine rheumatoid arthritis.

To determine whether canine rheumatoid arthritis (CRA) is associated with dog MHC (DLA-DRB1) alleles which contain the QRRAA/RKRAA conserved third hypervariable region (3HVR) sequence, DNA samples were extracted from 61 dogs with clinically diagnosed small-joint polyarthritis and from 425 controls. Breed-matched controls were available for 41 cases. DLA-DRB1 genotypes were identified using molecular typing methods. Phenotype frequencies were compared between cases and controls and odds ratios with 95% confidence intervals calculated. Several DLA-DRB1 alleles were associated with increased risk for CRA: DLA-DRB1*002, DRB1*009, and DRB1*018. This was also observed for the presence of any shared epitope (SE)-bearing allele. The associations with DLA-DRB1*002 and the SE were maintained when only breed-matched cases and controls were compared. This study suggests that a conserved amino acid motif in the 3HVR present in some DRB1 alleles of both dogs and humans is associated with rheumatoid arthritis in both species.

Canine rheumatoid arthritis and inflammatory cytokines.

Bioassays were developed to detect canine pro-inflammatory cytokines. These enabled characterisation of these cytokines and their isoforms and provided means for their assay in the joints of dogs with different naturally occurring arthropathies. Canine IL-1 was detected by its induction of proliferation of D10(N4)M cell line, whilst IL-6 had a proliferative effect on B9 cell line. TNFalpha had a cytotoxic effect on WEHI 164 (13) cells. Partial purification of the cytokines was achieved by FPLC ion-exchange chromatography and two isoforms of IL-1 were shown, possibly corresponding to IL-1alpha and IL-1beta. TNFalpha only appeared as one isoform whereas IL-6 showed at least five isoforms, possibly corresponding to the other molecules in the IL-6 family, such as IL-11 and oncostatin M. Analysis of synovial fluids from dogs with osteoarthritis (OA) and rheumatoid arthritis (RA) showed that IL-1 and TNFalpha bioactivity was not readily detectable at increased levels in diseased joints but that IL-6 was significantly increased in both diseases. It is now important to determine the role of IL-6 in OA and RA in the dog, particularly in the induction of proteolytic enzymes which lead to cartilage loss.

Immunocytochemical demonstration of lymphocyte subsets and MHC class II antigen expression in synovial membranes from dogs with rheumatoid arthritis and degenerative joint disease.

The study describes the distribution of canine leucocyte antigens in synovial membrane biopsies from six dogs with canine rheumatoid arthritis (CRA) and from eight dogs with osteoarthritis (OA) secondary to spontaneous rupture of the cranial cruciate ligament (CCL) (n = 5) or patellar luxation (n = 3). Synovial membranes from five dogs without evidence of joint lesions were used as control tissues. In the subsynovium of dogs with normal joints CD5+, CD4+, CD8+ and alpha beta TCR+ lymphocytes were present only in low numbers. With monoclonal antibody (mAb) to MHC class II antigen, either none or up to 20-30% of synovial lining cells were immunoreactive. Furthermore, scattered MHCII+ stromal cells were seen in the deeper subsynovial layer. In synovial membrane biopsies from dogs with CRA numerous diffusely and perivascularly distributed CD5+ lymphocytes were found in the subsynovium. CD4+ cells outnumbered CD8+ cells and were more numerous in the perivascular areas. In all the CRA cases examined, there were markedly higher numbers of alpha beta TCR+ cells compared with gamma delta TCR+ cells. With mAb to CD21, low numbers of immunoreactive lymphocytes were demonstrated. In all the CRA cases, a marked increase of MHC class II antigen expression was noted. In the majority of samples, 50% or more than 90% of the synovial lining cells were strongly MHC class II+. Throughout the subsynovial layer there were numerous MHC class II+ cells and included those with dendritic morphology and inflammatory mononuclear cells. Furthermore, marked perivascular immunoreactivity for MHC class II antigen was found. In biopsies from dogs with OA, there were markedly lower numbers of subsynovial CD5+, CD4+ and CD8+ lymphocytes. T-cells were mainly diffusely distributed. In three of the eight OA dogs examined, there was an increased percentage of synovial lining cells expressing MHC class II. The majority of OA cases had subsynovial major histocompatibility complex (MHC) class II+ cells with a dendritic morphology.

Matrix metalloproteinases 2 and 9 in canine rheumatoid arthritis.

Matrix metalloproteinases (MMPs) are considered important mediators of tissue damage in joint diseases. The levels of MMPs 2 and 9 were measured in samples of synovial fluid from 20 joints in seven dogs with rheumatoid arthritis by gelatin zymography. The results were compared with the actual gelatinolytic activity of the fluid measured in a gelatin-degradation ELISA. The gelatinolytic activity in synovial fluid from arthritic joints was markedly greater than that in fluid from disease-free joints. The zymographic activity attributable to MMP-9 (identified by Western blotting) was absent from synovial fluid from control joints but prominent in fluid from arthritic joints, and in these joints the presence of a 75 kDa form of MMP-9 was correlated with the gelatinolytic activity of the fluid measured by the ELISA (r = 0.81, P < 0.05). Synovial fluid from one dog with rheumatoid arthritis was examined before and after treatment with corticosteroids. After treatment its zymographic pattern had returned to normal.

Molecular cloning and cartilage gene expression of equine stromelysin 1 (matrix metalloproteinase 3).

OBJECTIVE: To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage. SAMPLES AND PROCEDURE: Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was assessed in normal and arthritic cartilage and synovium by northern blotting. Interleukin 1 (IL-1) regulation of stromelysin transcriptional activity in articular chondrocytes cultured in the presence of 0, 20, and 50 ng of IL-1 alpha/ml was assessed by northern blotting of total RNA isolated from the cell layer and probed with 32P-labeled stromelysin cDNA. RESULTS: 4 overlapping clones provided the full-length cDNA sequence of equine stromelysin, including portions of untranslated 5' and 3' regions, and the entire translated portion coding for the stromelysin prepropeptide. The coding region of 1,431 base pairs was well conserved between species, with 86, 83, and 78% sequence homology to that of human, rabbit, and mouse stromelysin, respectively. Predicted amino-acid (AA) sequence data indicated highly conserved features. Comparison of the equine AA sequence revealed 89, 88, and 84% homology to the AA structure in human, rabbit, and mouse stromelysin, respectively. Minimal stromelysin mRNA expression was evident in normal cartilage and synovium, and increased expression was evident in arthritic cartilage. Marked dose-dependent up-regulation of stromelysin transcriptional activity was evident in chondrocyte cultures exposed to 20 and 50 ng of IL-1/ml. CONCLUSIONS: Stromelysin DNA sequence in horses is similar to that in people and rodents. Constitutive stromelysin message amounts in normal cartilage and synovium are low, but considerably increased in arthritic cartilage and in chondrocytes exposed to IL-1.

Antibodies to heat shock proteins in dogs with rheumatoid arthritis and systemic lupus erythematosus.

Antibodies to heat shock proteins of the 65 kDa group were demonstrated in canine sera and synovial fluid. This paper reports these antibody measurements in three groups of dogs with joint disease and compares them with those of a control population. Dogs with rheumatoid arthritis (RA) showed higher anti-heat shock proteins (HSP) antibody levels, in both their sera and synovial fluids, compared to the control dogs and these antibodies were predominantly of the IgG and IgM class; there was a significant correlation between IgM anti-HSP65 and IgM-rheumatoid factors. There was also a significant correlation between anti-HSP65 and antibodies to canine distemper virus, but only of the IgM class and the relevance of these antibodies to the overall pathogenesis of canine RA and, in particular, to the presence of canine distemper virus within the joint, are discussed.

[Discospondylitis and immune-mediated polyarthritis in a Bernese mountain-dog]. / Discospondylitis en immuungemedieerde polyarthritis bij een Berner Sennenhond.

In this case report we describe the clinical picture and treatment of a Bernese mountain dog with discospondylitis and a presumably reactive immune-mediated polyarthritis. The clinical signs consisted of apathy, fever, anorexia, and a stiff gait. The diagnosis was based on the typical radiographic signs of discospondylitis and the cytology of the synovial fluid. The dog was treated with a broad-spectrum antibiotic for 6 weeks and thereafter with a synthetic glucocorticoid for the polyarthritis. Five months after cessation of therapy, the dog was free from the initial signs.

Hyaluronan in canine arthropathies.

Soluble hyaluronan (HA), which has been considered as a marker for joint disease in man, was measured in serum and synovial fluid (SF) from dogs with osteoarthritis (OA), rheumatoid arthritis (RA), and rupture of the cranial cruciate ligament (CCL) and from normal dogs (control). Dogs with OA and RA had significantly increased serum HA (P < 0.001) and decreased synovial fluid HA (P < 0.001), as did dogs with CCL rupture (serum, P < 0.05; synovial fluid, P < 0.005). In OA, HA was lower in the SF from the affected joint than in that from the clinically normal (inactive) contralateral joint; no such difference was seen in dogs with CCL rupture. Dogs with liver disease (portocaval shunts, viral infectious hepatitis, metastatic neoplasm and disease secondary to diabetes mellitus) had increased serum HA concentrations (P < 0.001). There was a significant overlap of HA values in the diseased and normal dogs. Therefore, it is unlikely that the measurement of this cartilage breakdown product would be of value for diagnosis or prognosis in canine arthropathies.

Rheumatoid arthritis.

Much has been learned about the interactions of immunologic events and the development of musculoskeletal disease. Much more needs to be learned. The partnership of astute clinicians and investigators will enhance this learning process and eventually benefit the health and well being of our patients, and perhaps, humankind.