Silica-coated calcium phosphate nanoparticles for gene silencing of NF-κB p65 by siRNA and their impact on cellular players of inflammation.

The transcription factor NF-κB and its signaling cascade both play key roles in all inflammatory processes. The most critical member of the NF-κB transcription factor family is p65. We investigated the role of cationic silica-coated calcium phosphate nanoparticles (spherical, diameter by SEM 50-60 nm; zeta potential about +26 mV; stabilized by polyethyleneimine) carrying encapsulated siRNA against NF-κB p65 and their influence on inflamed cells. The nanoparticles were taken up by cells of the blood compartment involved in the inflammatory response, particularly by monocytes, and to a lesser extent by endothelial cells and B-cells, but not by T-cells. The particles were found in endolysosomes where they were dissolved at low pH and released the siRNA into the cytoplasm. This was confirmed by dissolution experiments of model nanoparticles in simulated endolysosomal medium (pH 4.7) and by intracellular co-localization studies of double-labeled nanoparticles (using a negatively charged model peptide for siRNA). The encapsulated functional siRNA reverted the p65 gene and protein expression in inflamed monocytes, the main cells in immune response and surveillance, almost back to the non-inflammatory condition. Additionally, the nanoparticles suppressed the pro-inflammatory cytokine expression profiles (TNF-α, IL-6, IFN-ß) in inflamed J774A.1 monocytes. Taken together, such nanoparticles can be applied for the treatment of inflammatory diseases.

Chemoradiotherapy by intensity-modulated radiation therapy with simultaneous integrated boost in locally advanced or oligometastatic non-small-cell lung cancer-a two center experience.

PURPOSE: Integrating moderate hypofractionation to the macroscopic tumor with elective nodal irradiation while sparing the organs at risk (OAR) in chemoradiotherapy of locally advanced non-small-cell lung cancer. METHODS: From 2010-2018, treatment, patient and tumor characteristics of 138 patients from two radiation therapy centers were assessed. Chemoradiotherapy by intensity-modulated radiation therapy (IMRT) with a simultaneous integrated boost (SIB) to the primary tumor and macroscopic lymph node metastases was used. RESULTS: A total of 124 (90%) patients received concurrent chemotherapy. 106 (76%) patients had UICC (Union for International Cancer Control) stage ≥IIIB and 21 (15%) patients had an oligometastatic disease (UICC stage IV). Median SIB and elective total dose was 61.6 and 50.4 Gy in 28 fractions, respectively. Furthermore, 64 patients (46%) had an additional sequential boost to the primary tumor after the SIB-IMRT main series: median 6.6 Gy in median 3 fractions. The median cumulative mean lung dose was 15.6 Gy (range 6.2-29.5 Gy). Median follow-up and radiological follow-up for all patients was 18.0 months (range 0.6-86.9) and 16.0 months (range 0.2-86.9), respectively. Actuarial local control rates at 1, 2 and 3 years were 80.4, 68.4 and 57.8%. Median overall survival and progression-free survival was 30.0 months (95% confidence interval [CI] 23.5-36.4) and 12.1 months (95% CI 8.2-16.0), respectively. Treatment-related toxicity was moderate. Radiation-induced pneumonitis grade 2 and grade 3 occurred in 13 (9.8%) and 3 (2.3%) patients. CONCLUSIONS: Chemoradiotherapy using SIB-IMRT showed promising local tumor control rates and acceptable toxicity in patients with locally advanced and in part oligometastatic lung cancer. The SIB concept, resulting in a relatively low mean lung dose, was associated with low numbers of clinically relevant pneumonitis. The overall survival appears promising in the presence of a majority of patients with UICC stage ≥IIIB disease.

Functional Outcomes of Revision Osteosynthesis after Failure of Surgical Treatment of Patellar Fractures.

Patellar fractures account for approximately 1% of all skeletal fractures. These fractures are rare; however, because of the crucial function of the patella in the extensor mechanism of the knee, they may lead to serious impairment. New data are revealing functional impairment remains common even with improved surgical techniques. The aim of this study was to assess the functional outcomes of patients after revision surgery in cases of secondary fracture dislocation or persistent articular incongruity. This study included 16 patients with a mean age of 51.8 years (range: 16-85 years) with a mean follow-up of 35.1 months. According to the AO/OTA classification, 15 patients had a C-type fracture, including 10 patients with C3 fracture. Thirteen patients were initially treated with tension band wiring via K-wires or cannulated screws. Revision surgery was performed because of persistent articular incongruity in five patients and secondary fracture dislocation or refracture in 11 patients. We analyzed pain (visual analog scale [0-10]), satisfaction, range of motion (ROM), Böstman's score, Lysholm's score, and knee injury and osteoarthritis outcome score (KOOS) after revision surgery and could extract follow-up data from 15 patients. Mean pain score at rest was 0.57 (range: 0-3.5) and on exertion 2.79 (range: 0-8). The measurement of the ROM of the affected knee compared with that of the opposite knee revealed complete extension. Mean flexion was 123 degrees, in the corresponding knee it was 136 degrees (p = 0.05). The mean postoperative Böstman's and Lysholm's scores were 25.11 (good, maximum: 30) and 78.67 (moderate, maximum: 100), respectively. KOOS was as follows: symptoms, 66.8 points; pain, 77.55 points; activity of daily living (ADL), 75.67 points; and quality of life, 56.25 points. The results of this study suggested that early revision surgery after failure of primary osteosynthesis with secondary anatomic reconstruction and good radiological results leads to satisfactory functional outcomes with persistent functional deficits.

5-Azacytidine modulates CpG methylation levels of EZH2 and NOTCH1 in myelodysplastic syndromes.

PURPOSE: Molecular mechanisms of response to hypomethylating agents in patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) still remain largely unknown. Therefore, the effects of 5-Azacytidine (Aza) on clonal architecture and DNA methylation were investigated in this study. METHODS: Using next-generation sequencing (NGS), 30 myeloid leukemia-associated genes were analyzed in 15 MDS/CMML patients with excellent response to Aza. Effects on methylation levels were analyzed by quantitative methylation analysis using pyrosequencing for the global methylation marker LINE-1 in patients and myeloid cell lines. Various myeloid cell lines and a healthy cohort were screened for methylation levels in 23 genes. Selected targets were verified on the MDS/CMML cohort. RESULTS: The study presented here showed a stable variant allele frequency and stable global methylation levels in responding patients. A significant demethylation of EZH2 and NOTCH1 was revealed in patients with Aza response. CONCLUSIONS: A response to Aza is not associated with eradication of malignant clones, but rather with a stabilization of the clonal architecture. We suggest changes in CpG methylation levels of EZH2 and NOTCH1 as potential targets of epigenetic response to Aza treatment which may also serve as useful biomarkers after clinical evaluation.

Iron overload and non-alcoholic fatty liver disease.

Approximately one third of patients with non-alcoholic fatty liver disease (NAFLD) show signs of disturbed iron homeostasis as indicated by elevated serum ferritin with normal or mildly elevated transferrin saturation. Mild hepatic iron deposition is the typical histological finding in these subjects and the term "dysmetabolic iron overload syndrome (DIOS)" is now used to describe this syndrome. From a clinical point of view, excess iron likely aggravates the natural course of NAFLD with regard to liver-related endpoints and extrahepatic disease complications although sound evidence is still lacking. The detrimental effect of iron is attributed to its capability to catalyse the formation of toxic hydroxyl radicals resulting in cellular damage. Conversely, reduction of body iron restores insulin sensitivity, and epidemiological evidence suggests that it delays the onset of complications such as T2DM, cardiovascular disease and also advanced liver disease. Iron accumulation in NAFLD is mainly due to inhibition of iron mobilisation from hepatocytes and Kupffer cells. Impaired iron export is related to inflammation and metabolic derangements that appear to impact on iron regulators, such as hepcidin, ferroportin and to a lesser degree on transferrin receptor, ferritin or copper. This review summarizes the current understanding of the role of iron in NAFLD.