Osteo-immunological impact of radon spa treatment: due to radon or spa alone? Results from the prospective, thermal bath placebo-controlled RAD-ON02 trial.

Musculoskeletal disorders (MSDs) are associated with pain and lead to reduced mobility and quality of life for patients. Radon therapy is used as alternative or complementary to pharmaceutical treatments. According to previous reports, radon spa leads to analgesic and anti-inflammatory effects, but the cellular and molecular mechanisms are widely unknown. A previous study (RAD-ON01) revealed, that bone erosion markers like collagen fragments (C-terminal telopeptide, CTX) are reduced after radon spa treatment in serum of patients with degenerative MSDs. Within the scope of the prospective, placebo-controlled RAD-ON02 trial presented here, we analyzed the influence of radon and thermal spa treatment on osteoclastogenesis. From patient blood, we isolate monocytes, seeded them on bone slices and differentiated them in the presence of growth factors into mature osteoclasts (mOCs). Subsequent analysis showed a smaller fraction of mOCs after both treatments, which was even smaller after radon spa treatment. A significantly reduced resorbed area on bone slices reflects this result. Only after radon spa treatment, we detected in the serum of patients a significant decrease of receptor activator of NF-κB ligand (RANKL), which indicates reduced differentiation of OCs. However, other markers for bone resorption (CTX) and bone formation (OPG, OCN) were not altered after both treatments. Adipokines, such as visfatin and leptin that play a role in some MSD-types by affecting osteoclastogenesis, were not changed after both treatments. Further, also immune cells have an influence on osteoclastogenesis, by inhibiting and promoting terminal differentiation and activation of OCs, respectively. After radon treatment, the fraction of Treg cells was significantly increased, whereas Th17 cells were not altered. Overall, we observed that both treatments had an influence on osteoclastogenesis and bone resorption. Moreover, radon spa treatment affected the Treg cell population as well as the Th17/Treg ratio were affected, pointing toward a contribution of the immune system after radon spa. These data obtained from patients enrolled in the RAD-ON02 trial indicate that radon is not alone responsible for the effects on bone metabolism, even though they are more pronounced after radon compared to thermal spa treatment.

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure.

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.

Radon Improves Clinical Response in an Animal Model of Rheumatoid Arthritis Accompanied by Increased Numbers of Peripheral Blood B Cells and Interleukin-5 Concentration.

Radon treatment is used as an established therapy option in chronic painful inflammatory diseases. While analgesic effects are well described, little is known about the underlying molecular effects. Among the suspected mechanisms are modulations of the anti-oxidative and the immune system. Therefore, we aimed for the first time to examine the beneficial effects of radon exposure on clinical outcome as well as the underlying mechanisms by utilizing a holistic approach in a controlled environment of a radon chamber with an animal model: K/BxN serum-induced arthritic mice as well as isolated cells were exposed to sham or radon irradiation. The effects on the anti-oxidative and the immune system were analyzed by flow-cytometry, qPCR or ELISA. We found a significantly improved clinical disease progression score in the mice, alongside significant increase of peripheral blood B cells and IL-5. No significant alterations were visible in the anti-oxidative system or regarding cell death. We conclude that neither cell death nor anti-oxidative systems are responsible for the beneficial effects of radon exposure in our preclinical model. Rather, radon slightly affects the immune system. However, more research is still needed in order to fully understand radon-mediated effects and to carry out reasonable risk-benefit considerations.

ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells.

Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose-effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0-2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source.

A small viral potassium ion channel with an inherent inward rectification.

Some algal viruses have coding sequences for proteins with structural and functional characteristics of pore modules of complex K+ channels. Here we exploit the structural diversity among these channel orthologs to discover new basic principles of structure/function correlates in K+ channels. The analysis of three similar K+ channels with ≤ 86 amino acids (AA) shows that one channel (Kmpv1) generates an ohmic conductance in HEK293 cells while the other two (KmpvSP1, KmpvPL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K+ driving force. Reconstitution of KmpvSP1 and KmpvPL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K+ reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be an inherent property of the channel. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations making the rectifier similar to the ohmic conducting channel. The results of these experiments exclude the pore with pore helix and selectivity filter as playing a role in rectification. The insensitivity of the rectifier to point mutations suggests that tertiary or quaternary structural interactions between the transmembrane domains are responsible for this type of gating.