NOX2 and NOX4 expression in monocytes and macrophages-extracellular vesicles in signalling and therapeutics.

Extracellular vesicles (EVs) are a type of cytoplasmic vesicles secreted by a variety of cells. EVs originating from cells have been known to participate in cell communication, antigen presentation, immune cell activation, tolerance induction, etc. These EVs can also carry the active form of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Hydrogen (NADPH) oxidase, which is very essential for the production of reactive oxygen species (ROS) and that can then modulate processes such as cell regeneration. The aim of this study is to characterize the EVs isolated from U-937 and THP-1 cells, identify the NADPH oxidase (NOX) isoforms, and to determine whether EVs can modulate NOX4 and NOX2 in monocytes and macrophages. In our study, isolated EVs of U-937 were characterized using dynamic light scattering (DLS) spectroscopy and immunoblotting. The results showed that the exogenous addition of differentiation agents (either phorbol 12-myristate 13-acetate (PMA) or ascorbic acid) or the supplementation of EVs used in the study did not cause any stress leading to alterations in cell proliferation and viability. In cells co-cultured with EVs for 72 h, strong suppression of NOX4 and NOX2 is evident when monocytes transform into macrophagic cells. We also observed lower levels of oxidative stress measured using immunoblotting and electron paramagnetic resonance spectroscopy under the EVs co-cultured condition, which also indicates that EVs might contribute significantly by acting as an antioxidant source, which agrees with previous studies that hypothesized the role of EVs in therapeutics. Therefore, our results provide evidence for NOX regulation by EVs in addition to its role as an antioxidant cargo.

Differential effects of ascorbic acid on monocytic cell morphology and protein modification: Shifting from pro-oxidative to antioxidant properties.

In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.

Total ablative stereotactic body radiotherapy to primary and oligometastatic renal cell carcinoma in medically inoperable cases: An institutional analysis.

Introduction: Stereotactic body radiotherapy (SBRT) has been found to be an effective and safe modality with excellent oncological outcome in medically inoperable primary renal cell carcinoma (RCC) and oligometastases. There is scarcity of data on the synchronous delivery of SBRT to primary and oligometastatic RCC in patients unfit for nephrectomy. Here, we report the findings of a retrospective study of prospectively collected data on "total ablative SBRT." Methods: Oligometastatic RCC patients with intact primary tumors were enrolled between May 2021 and June 2022. SBRT was synchronously delivered to the primary tumor and metastases. Demographics, treatment, oncologic outcomes, and toxicity were assessed. Kaplan-Meier estimates were generated for oncologic outcomes. The primary endpoint of this study was feasibility and tolerability. Results: Eleven patients were enrolled between May 2021 and June 2022. One patient died at 2 months after SBRT due to viral pneumonitis (possibly COVID pneumonia). Nine patients (82%) had metastatic disease, while 2 (18%) were stage II. The average maximal diameter of primary was 68.7 mm (range, 23-128 mm). The SBRT doses for primary and metastasis ranged from 40 to 55 Gray (Gy) in 5 to 7 fractions and 22 to 40Gy in 2 to 5 fractions, respectively. The median follow-up period was 10.5 months (Range: 4-15 months). Response assessment was available for ten patients. Local control, marginal control, regional control and initial oligometastatic control (OMC) rates were 100%. OMC declined to 87.5% as one patient had recurrence in irradiated subcarinal lymphnode at 7 months. The metastatic control rate was 80% and loco-regional progression-free survival was 8 months (range, 4-15 months). Toxicities were minimal and manageable. At the last follow-up, 7 of 11 patients were alive with an overall survival of 63.5%. Six patients received systemic therapy after SBRT. Conclusions: Synchronous delivery of SBRT to primary and oligometastatic sites in patients unfit for nephrectomy was feasible and tolerable with good locoregional control. The total ablative SBRT strategy needs to be explored in similar cohorts.

Imaging and Characterization of Oxidative Protein Modifications in Skin.

Skin plays an important role in protection, metabolism, thermoregulation, sensation, and excretion whilst being consistently exposed to environmental aggression, including biotic and abiotic stresses. During the generation of oxidative stress in the skin, the epidermal and dermal cells are generally regarded as the most affected regions. The participation of reactive oxygen species (ROS) as a result of environmental fluctuations has been experimentally proven by several researchers and is well known to contribute to ultra-weak photon emission via the oxidation of biomolecules (lipids, proteins, and nucleic acids). More recently, ultra-weak photon emission detection techniques have been introduced to investigate the conditions of oxidative stress in various living systems in in vivo, ex vivo and in vitro studies. Research into two-dimensional photon imaging is drawing growing attention because of its application as a non-invasive tool. We monitored spontaneous and stress-induced ultra-weak photon emission under the exogenous application of a Fenton reagent. The results showed a marked difference in the ultra-weak photon emission. Overall, these results suggest that triplet carbonyl (3C=O∗) and singlet oxygen (1O2) are the final emitters. Furthermore, the formation of oxidatively modified protein adducts and protein carbonyl formation upon treatment with hydrogen peroxide (H2O2) were observed using an immunoblotting assay. The results from this study broaden our understanding of the mechanism of the generation of ROS in skin layers and the formation/contribution of various excited species can be used as tools to determine the physiological state of the organism.

Bioactive Compounds and Their Impact on Protein Modification in Human Cells.

Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.