BackgroundExosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.AimTherefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).Methods and ResultsExosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.ConclusionHere we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.

The aryl hydrocarbon receptor (AHR) is a cytoplasmic transcription factor, which plays an essential role in the xenobiotic metabolism in a wide variety of cells. The AHR gene is evolutionarily conserved and it has a central role not only in the differentiation and maturation of many tissues, but also in the toxicological metabolism of the cell by the activation of metabolizing enzymes. Several lines of evidence support that both AHR agonists and antagonists have profound immunological effects; and recently, the AHR has been implicated in antibacterial host defense. According to recent studies, the AHR is essential for the differentiation and activation of T helper 17 (Th17) cells. It is well known that Th17 cells have a central role in the development of inflammation, which is crucial in the defense against pathogens. In addition, Th17 cells play a major role in the pathogenesis of several autoimmune diseases such as rheumatoid arthritis. Therefore, the AHR may provide connection between the environmental chemicals, the immune regulation, and autoimmunity. In the present review, we summarize the role of the AHR in the Th17 cell functions.

Although Src-like adaptor proteins (SLAP-1 and SLAP-2) were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins.

BackgroundThe T-helper 17 (Th17) cells have a prominent role in inflammation as well as in bone and join destruction in both rheumatoid and psoriatic arthritis (RA and PsA). Here, we studied Th17 cell differentiation in RA and PsA.MethodsBlood samples from healthy donors, RA and PsA patients were collected. CD45RO− (naive) and CD45RO+ (memory) T cells were isolated from peripherial blood mononuclear cell by magnetic separation. Naive T cells were stimulated with anti-CD3, anti-CD28, and goat anti-mouse IgG antibodies and treated with transforming grow factor beta, interleukin (IL)-6, IL-1β, and IL-23 cytokines and also with anti-IL-4 antibody. IL-17A and IL-22 production were measured by enzyme linked immunosorbent assay, RORC, and T-box 21 (TBX21) expression were analyzed by quantitative polymerase chain reaction and flow cytometry. C-C chemokine receptor 6 (CCR6), CCR4, and C-X-C motif chemokine receptor 3 expression were determined by flow cytometry. Cell viability was monitored by impedance-based cell analyzer (CASY-TT).ResultsRORC, TBX21, CCR6, and CCR4 expression of memory T cells of healthy individuals (but not RA or PsA patients) were increased (p < 0.01; p < 0.001; p < 0.05; p < 0.05, respectively) compared to the naive cells. Cytokine-induced IL-17A production was different in both RA and PsA patients when compared to healthy donors (p = 0.0000026 and p = 0.0001047, respectively). By contrast, significant differences in IL-22 production were observed only between RA versus healthy or RA versus PsA patients (p = 0.000006; p = 0.0013454, respectively), but not between healthy donors versus PsA patients.ConclusionThe naive CD4 T-lymphocytes are predisposed to differentiate into Th17 cells and the in vitro Th17 cell differentiation is profoundly altered in both RA and PsA.