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
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
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.
The treatment of chronic inflammatory and degenerative diseases by low dose radiation therapy (LDRT) is promising especially for patients who were refractory for classical therapies. LDRT aims to ...reduce pain of patients and to increase their mobility. Although LDRT has been applied since the late 19th century, the immunological mechanisms remain elusive. Within the prospective IMMO-LDRT01 trial (NCT02653079) the effects of LDRT on the peripheral blood immune status, as well as on pain and life quality of patients have been analyzed. Blood is taken before and after every serial irradiation with a single dose per fraction of 0.5Gy, as well as during follow-up appointments in order to determine a detailed longitudinal immune status by multicolor flow cytometry. Here, we report the results of an interim analysis of 125 patients, representing half the number of patients to be recruited. LDRT significantly improved patients' pain levels and induced distinct systemic immune modulations. While the total number of leukocytes remained unchanged in the peripheral blood, LDRT induced a slight reduction of eosinophils, basophils and plasmacytoid dendritic cells and an increase of B cells. Furthermore, activated immune cells were decreased following LDRT. Especially cells of the monocytic lineage correlated to LDRT-induced improvements of clinical symptoms, qualifying these immune cells as predictive biomarkers for the therapeutic success. We conclude that LDRT improves pain of the patients by inducing systemic immune modulations and that immune biomarkers could be defined for prediction by improved patient stratification in the future.
PurposeThe first aim of the trial is to study feasibility of combined programmed death protein ligand 1/cytotoxic T-lymphocyte-associated protein 4 inhibition concomitant to radiotherapy. In ...addition, efficacy of the entire treatment scheme consisting of induction chemoimmunotherapy followed by chemotherapy-free radioimmunotherapy (RIT) after intratumoral CD8 +immune cell-based patient selection will be analyzed.MethodsPatients with stage III–IVB head and neck squamous cell carcinoma were eligible for this multicenter phase II trial. Treatment consisted of a single cycle of cisplatin 30 mg/m² days 1–3, docetaxel 75 mg/m² day 1, durvalumab 1500 mg fix dose day 5 and tremelimumab 75 mg fix dose day 5. Patients with increased intratumoral CD8 +immune cell density or pathological complete response (pCR) in the rebiopsy entered RIT up to a total dose of 70 Gy. Patients received further three cycles of durvalumab/tremelimumab followed by eight cycles of durvalumab mono (every 4 weeks). The intended treatment for patients not meeting these criteria was standard radiochemotherapy outside the trial. Primary endpoint was a feasibility rate of patients entering RIT to receive treatment until at least cycle 6 of immunotherapy of ≥80%.ResultsBetween September 2018 and May 2020, 80 patients were enrolled (one excluded). Out of these, 23 patients had human papilloma virus (HPV)-positive oropharyngeal cancer. Median follow-up was 17.2 months. After induction chemoimmunotherapy 41 patients had pCR and 31 had increased intratumoral CD8 +immune cells. Of 60 patients entering RIT (primary endpoint cohort), 10 experienced imiting toxic (mainly hepatitis) and four discontinued for other reasons, resulting in a feasibility rate of 82%. The RIT cohort (n=60) had a progression-free survival (PFS) rate at one and 2 years of 78% and 72%, respectively, and an overall survival rate at one and 2 years of 90% and 84%, respectively. Patients with HPV-positive oropharyngeal cancers had greater benefit from RIT with a 2-year PFS rate of 94% compared with 64% for HPV-negative oropharyngeal cancers and other locations. In the entire study cohort (n=79) the 2-year PFS rate was 68% (91% for HPV-positive oropharynx vs 59% for others). Toxicity grade 3–4 mainly consisted of dysphagia (53%), leukopenia (52%) and infections (32%).ConclusionsThe trial met the primary endpoint feasibility of RIT. Induction chemo-immunotherapy followed by chemotherapy-free RIT after intratumoral CD8 +immune cell-based patient selection has promising PFS.Trial registration numberThe trial was registered with ClinicalTrials.gov (identifier: NCT03426657). The trial was conducted as investigator-sponsored trial (IST).
Osteoarthritis (OA) is one of the most common and socioeconomically relevant diseases, with rising incidence and prevalence especially with regard to an ageing population in the Western world. Over ...the decades, the scientific perception of OA has shifted from a simple degeneration of cartilage and bone to a multifactorial disease involving various cell types and immunomodulatory factors. Despite a wide range of conventional treatment modalities available, a significant proportion of patients remain treatment refractory. Low-dose radiotherapy (LDRT) has been used for decades in the treatment of patients with inflammatory and/or degenerative diseases and has proven a viable option even in cohorts of patients with a rather poor prognosis. While its justification mainly derives from a vast body of empirical evidence, prospective randomized trials have until now failed to prove the effectiveness of LDRT. Nevertheless, over the decades, adaptions of LDRT treatment modalities have evolved using lower dosages with establishment of different treatment schedules for which definitive clinical proof is still pending. Preclinical research has revealed that the immune system is modulated by LDRT and very recently osteoimmunological mechanisms have been described. Future studies and investigations further elucidating the underlying mechanisms are an essential key to clarify the optimal patient stratification and treatment procedure, considering the patients’ inflammatory status, age, and sex. The present review aims not only to present clinical and preclinical knowledge about the mechanistic and beneficial effects of LDRT, but also to emphasize topics that will need to be addressed in future studies. Further, a concise overview of the current status of the underlying radiobiological knowledge of LDRT for clinicians is given, while seeking to stimulate further translational research.
Radiotherapy (RT) of the brain is a common treatment for patients with high-grade gliomas and brain metastases. It has previously been shown that reactivation of cytomegalovirus (CMV) frequently ...occurs during RT of the brain. This causes neurological decline, demands antiviral treatment, and is associated with a worse prognosis. CMV-specific T cells are characterized by a differentiated effector memory phenotype and CD45RA+ CCR7- effector memory T (T
) cells were shown to be enriched in CMV seropositive individuals. In this study, we investigated the distribution of T
cells and their subsets in the peripheral blood of healthy donors and, for the first time, prospectively within the scope of the prospective Glio-CMV-01 clinical trial of patients with high-grade glioma and brain metastases during radiation therapy as a potential predictive marker. First, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of T
cells in a longitudinal manner. The CMV serostatus and age were considered as influencing factors. We revealed that patients who had a reactivation of CMV have significantly higher amounts of CD8+ T
cells. Further, the distribution of the subsets of T
cells based on the expression of CD27, CD28, and CD57 is highly dependent on the CMV serostatus. We conclude that the percentage of CD8+ T
cells out of all CD8+ T cells has the potential to serve as a biomarker for predicting the risk of CMV reactivation during RT of the brain. Furthermore, this study highlights the importance of taking the CMV serostatus into account when analyzing T
cells and their subsets.
Recent improvements in the flow cytometry technology allow the determination of the general immune status through the development of multicolor immunofluorescence panels. The one-tube multicolor flow ...cytometry assay (OTMA) that is presented here identifies 20 different, clinically relevant immune cell subsets and three common activation markers. Thereby, a comprehensive immune status that covers all major immune cells is easily obtained.The assay is suitable for every common three lasers and 10 color flow cytometer and includes the application of 15 different antibodies. Furthermore, the assay requires only 100 μL of EDTA-treated whole-blood and less than 40 min for sample preparation. By being easily adaptable to individual requirements and by additionally determining absolute cell counts, the assay is well-suited for translational research in clinical trials.
Supplementation of standard cancer therapies (radiotherapy, chemotherapy, surgery) with immunotherapies has revolutionized cancer treatment. In order to include recent improvements of multimodal ...therapies into clinical routine, knowledge about the immune status, the immune dynamics and the detailed composition and activation of patient's immune system is required. The here presented single-tube multicolor flow cytometry assay allows the discrimination of 20 clinically relevant immune cell subsets and their activation status in peripheral whole blood. It includes 15 different antibodies and can be established on a common 3 laser and 10 color flow-cytometer. Furthermore, this assay is easy to set-up and to perform as well as fast with only 40min of sample preparation time. Moreover, only 100μL of whole blood are sufficient for this precise determination of the individual immune status. It is already applied in translational programs of clinical studies and trials and can further be adapted for future ones.
Nowadays, ionizing radiation is applied clinically for the treatment of various disorders. High doses of ionizing radiation are used in tumor therapy to particularly achieve local tumor control. On ...the other hand, low doses of ionizing radiation are well known for the induction of pain-relieving effects in chronic degenerative and inflammatory diseases. Apart from the targeted effects of ionizing radiation such as the induction of cell death, it is eminent today that ionizing radiation is also able to modulate the immune system. In this context, low and high doses have opposite effects, as low doses can ameliorate inflammatory processes, while high doses are able to stimulate immune responses and inflammation. Currently, therapeutic concepts aim to integrate and utilize the immunomodulatory effects of radiation to improve patient outcomes. Thus, a deeper understanding of the longitudinal immunological changes in patients during radiotherapy is beneficial to optimize these therapeutic concepts, as well as to unravel the modes of action and to define biomarkers.Hence, we perform a flow cytometry-based immunophenotyping assay in our laboratory that allows to distinguish more than 20 different immune cell types and their respective activation status from peripheral blood in order to analyze the immune status of patients in clinical trials. In parallel, we collect serum and plasma for the quantification of inflammatory mediators such as pro- and anti-inflammatory cytokines. Within the scope of this work, we longitudinally monitored and analyzed the immune status in three independent patient studies together with the analysis of different clinical parameters.Our first goal was to unravel the immunological modes of action of low doses of ionizing radiation on the immune system. Thus, we initiated the IMMO-LDRT01 trial in 2015, which focuses on the analysis of the immune status of patients receiving low dose radiation therapy (X--rays) for chronic degenerative and inflammatory diseases. Even though a plethora of pre-clinical work exists on the modes of action of low dose radiation therapy, the immunomodulatory effects have never been analyzed in patients. In addition to immunological analyses, clinical data were collected on patient outcome and pain relief. We confirmed that low dose radiation therapy induces long-lasting analgesic effects and found sustained modulations of peripheral immune status. In detail, cells of the innate immune system, namely monocytic cells and granulocytes, were mainly altered. Further, low doses of X-rays showed a more prominent impact on the activation status of immune cells rather than their absolute counts. Certain cell types showed a significantly less active state after the application of low doses of ionizing radiation, as monitored by the expression of cell surface activation markers. The immunological modulation, as well as the clinical outcome were affected by the key clinical characteristics of the cohort, such as the underlying disease. Most strikingly, we observed that the monocytic cells correlated with the outcome of the patients, indicating that these cells will be promising predictive biomarkers for low dose radiation therapy in the future. The results of the IMMO-LDRT01 trial provide the basis for future randomized trials and deeper mechanistic analyses.Low doses of ionizing radiation are not only applied therapeutically in the form of low dose radiation therapy with X-rays, but also as radon spa therapy. Here, patients take serial baths in natural fountain water containing low concentrations of this radioactive noble gas.
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