Abstract. The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergenspecific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA.

Objective. To examine the interaction between synovial fibroblasts and macrophages in the context of cartilage degradation.Methods. An in vitro model of human cartilage degradation was used, in which purified populations of fibroblasts and macrophages were added to a radiolabeled cartilage disc. Cartilage destruction was measured by the percentage of radiolabel release.Results. Fibroblasts, obtained from either rheumatoid arthritis (RA) or osteoarthritis synovial tissue, could mediate cartilage degradation if cocultured with the U937 macrophage cell line. Skin and RA bone marrow fibroblasts had no degradative effect on cartilage. Fibroblast-macrophage contact was not required for cartilage degradation. Cartilage degradation by synovial fibroblasts was inhibited by antibodies to tumor necrosis factor a (TNFa), interleukin-lp (IL-lp), and IL-6. Cartilage degradation was almost completely abrogated by a combination of antibodies to TNFa and IL-1p. Contact between fibroblasts and cartilage was shown to be essential. Antibodies to CD44, but not to intercellular adhesion molecule 1, markedly inhibited cartilage degradation.Conclusion. TNFar, IL-lp, and IL-6 were involved in the activation of synovial fibroblasts to cause cartilage degradation. Cartilage degradation occurred only when fibroblasts were in contact with cartilage. CD44 was demonstrated to be involved in the fibroblastcartilage interaction. Rheumatoid arthritis (RA) is a disease in which immune system mediators evoke an inflammatory response within the joint resulting in cartilage and bone destruction. Evidence has been accumulated to indicate that the cartilage and bone destruction is mediated by synovial macrophages and fibroblasts as a consequence of metallaproteinase release (1-4). It has been suggested that fibroblasts and macrophages within the synovium orchestrate the destructive processes via a self-perpetuating autocrine/paracrine network (5). In support of this concept, fibroblasts and macrophages have an activated phenotype and secrete cytokines capable of paracrine interactions (i.e., interleukin-la [ILla], tumor necrosis factor a [TNFa], IL-6, and granulocyte-macrophage colony-stimulating factor) (6-10). In addition, it has been recently demonstrated that anti-TNFa therapy is effective in the treatment of RA To study the pathobiologic process within the RA joint, we reduced the inflammatory response to the level of its constitutive cellular elements. Using purified populations of cells, we were able to determine, in vitro, the cellular interactions that lead to joint destruction. We demonstrated a key, contact-dependent role for the synovial fibroblast in the destruction of cartilage, in synergy with 1 or more macrophage-generated cytokines. We also demonstrated that cartilage degradation in our assay system could be inhibited with antibodies directed to macrophage-or fibroblast-generated cytokines (i.e., anti-TNFa, anti-IL-la, and anti-IL-6) and with antibodies to adhesion molecule CD44. An additional inhibitory effect was demonstrated by usi...