pneumoniaespecific primers pairs detection. In conclusion, this study revealed the design of a PCR method for the detection of the MAM superantigen, and also determined the sensitivity and precision of the test. Results == The results of the PCR product sequencing showed the method has objective applicability and accuracy. The sensitivity of the PCR reaction for the reference DNA template was 1ng/ml. The PCR results assay of the 133 SF samples raveled that, 9. 7% and 22. 5% of them were positive for the MAM superantigen gene andMycoplasma pneumoniae (M. pneumoniae), respectively. == Conclusion == In this study, twoMycoplasmagenomes were detected with increased frequency in RA SF patients samples. This finding appears to be a promising instrument in the etiological diagnostic of RA patients and could also lead to improved treatment selection. Further research on the otherMycoplasmaspecies present in the SF of RA patients is essential. Keywords: Rheumatoid arthritis, Mycoplasma arthritidismitogen, Synovial fluid, Superantigen, PCR == INTRODUCTION == Mycoplasma arthritidis(M. arthritidis), as an animal pathogen is unique amongMycoplasmaspecies harboring the lysogenized bacteriophage MAV1, whose contribution to virulence, and production of the potentMycoplasma arthritidismitogen (MAM), that confers increased toxicity, lethality and arthritogenicity in experimental animals (1, 2). However , several reports mention that this bacterium was isolated from the synovial membranes, synovial fluid (SF), middle ear, eye, abscessed bone, abscessed ovary, oropharynx of wild and captive rats and also from joint fluid of non-human primates, including rhesus and wild boars (3-5). Reports on the isolation or molecular detection of this bacterium from human infections are unavailable. Based on the evidence, it has been postulated that superantigens might play a role in the autoimmune diseases and it was shown that the MAM superantigen can both trigger and exacerbate mouse autoimmune arthritis (6). Current interest in MAM MLT-747 superantigen relates to its ability to modulate the immune systemin vivothat may lead to the development of autoimmune diseases, such as rheumatoid arthritis (RA), respiratory, reproductive and inflammatory joint diseases in rodents (7-9). MAM has the potential for triggering autoimmune disease in mice (10). Other studies indicate MLT-747 TGFBR1 that MAM causes acute polyarthritis in rats and chronic proliferative arthritis in mice (11). In addition , macrophage activation byM. arthritidiscould play a significant role in the inflammatory response and induces toxicity, arthritis, and dermal necrosis in mice (12). The evidences indicating thatM. arthritidismay cause experimentally induced acute and chronic arthritis in animals has led several investigators to look for a similar potential in humans. Direct involvement of toll-like receptor (TLR2 and TLR4) in MAM binding and presentation to T cells was investigated and revealed that coexpression of TLR2 or TLR4 with the human leukocyte antigen-D related (HLA-DR) significantly increases MAM binding. The subsequent T cell activation may play an important role in the outcome of diseases induced byM. arthritidis(13, 14). A recent study demonstrated the reactivity of antibodies to MAM in the sera of RA patients (15). Furthermore, the results of another study proved that MAM has the capacity to induce proinflammatory cytokine transcription in monocytes via major histocompatibility complex (MHC) class II molecules. This phenomenon has been shown to activate one pathway of autoimmune diseases arthritis in rodents, which closely resembles human RA (16). However , cultivation and isolation ofM. arthritidisfrom clinical samples is more costly and time consuming. In addition , the role ofM. arthritidisin human arthritis remains MLT-747 unsolved. The aim of this study was to design PCR-based molecular methods for the detection of gene-encoded MAM superantigen in SF samples of patients with RA. == MATERIALS AND METHODS == == Standardization protocol == In this study, the complete sequences of MAM superantigen gene (Gene ID: 6418105) belongs toM. arthritidisstrain 158L3-1 with GenBank referenceNC_011025. 1was synthesized with as positive control in vector pGEM-B1 (Bioneer Corp, Daejeon, South Korea). The vector pGEM-B1 containing MAM superantigen gene (717bp) was transferred intoE. coliDH5 by the electroporation method (17). Ampicillin (100 g/ml) resistant transformants were selected and subjected to purification. This recombinant gene was used asM. arthritidisPCR positive control standard. In addition , aMycoplasma pneumoniae (M. pneumoniae)strain supplied by the Mycoplasma Reference Laboratory of the Razi.
