Major findings of the pilot study involving 21 critically ill patients during the week after admission to the critical care unit specialized for COVID-19 are presented. Fourteen patients have recovered, while seven passed away. There were no differences between them in respect to clinical or laboratory parameters monitored. However, protein adducts of the lipid peroxidation product 4-hydroxynonenal (HNE) were higher in the plasma of the deceased patients, while total antioxidant capacity was below the detection limit for the majority of sera samples in both groups. Moreover, levels of the HNE-protein adducts were constant in the plasma of the deceased patients, while in survivors, they have shown prominent and dynamic variations, suggesting that survivors had active oxidative stress response mechanisms reacting to COVID-19 aggression, which were not efficient in patients who died. Immunohistochemistry revealed the abundant presence of HNE-protein adducts in the lungs of deceased patients indicating that HNE is associated with the lethal outcome. It seems that HNE was spreading from the blood vessels more than being a consequence of pneumonia. Due to the limitations of the relatively small number of patients involved in this study, further research on HNE and antioxidants is needed. This might allow a better understanding of COVID-19 and options for utilizing antioxidants by personalized, integrative biomedicine approach to prevent the onset of HNE-mediated vitious circle of lipid peroxidation in patients with aggressive inflammatory diseases.

The activity principle of the mistletoe (Viscum album L.) phytotherapeutics could be considered as combined cytotoxic and "biological response modifying" activities (increasing host defense against cancer) that result from the activities of the plant lectins and the other biologically relevant substances. We found before that the aqueous extract Isorel, produced by Novipharm GmbH (Pörtschach, Austria) from the entire plant (planta tota) of fresh mistletoe under standardized conditions with bioassay validated batch consistency, can be valuable in experimental adjuvant cancer therapy increasing efficiency of cyclophosphamide chemotherapy. In current study we found that Isorel increases the reactivity of the tumor-bearing mice lymphocytes to the mitogens (ConA and LPS) in vitro, thus indicating its immune stimulating effects for the cancer-immunosuppressed lymphocytes. Moreover, Isorel inhibited the incorporation of 3H-labelled amino acids (protein synthesis) in various malignant cell lines. For the growth inhibition mostly higher MW components were responsible, although even less than 500 Da components were also active. We further analyzed the effects of drug application in vicinity of tumor (murine mammary carcinoma) and compared it with systemic effects. The animals carried mammary carcinoma in both hind limbs and were also injected with tumor cells i.v. to develop artificial lung metastases. Isorel was applied only at the right side (in the limb distal from the tumor) and caused persistent and almost complete inhibition of the tumor growth for 2/7 animals. Anticancer effects were less pronounced on the contralateral side tumors, although tumor growth rate was transiently reduced for some mice. Histology revealed that Isorel treatment, both at the side of tumor and systemically, increased the incidence of apoptosis and necrosis in the tumors, while reduction of mitosis was noticed only for the tumors in vicinity of the tumor exposed to Isorel. Finally, animals treated with Isorel had, on the average, three times less lung metastases than the controls. Thus, we conclude that both local and systemic effects of the application of Isorel could be of benefit for the tumor-bearing organism resulting in immunomodulation combined with tumor growth inhibition and reduction of metastases. According to the in vitro results, antitumorous effects could be the result not only of the mistletoe lectins and the other high MW factors, but also of the very low MW (< 500 Da) substances that deserve further analyses.

Oxidative stress has been implicated in pathophysiology of different human stress- and age-associated disorders, including osteoporosis for which antioxidants could be considered as therapeutic remedies as was suggested recently. The 1,4-dihydropyridine (DHP) derivatives are known for their pleiotropic activity, with some also acting as antioxidants. To find compounds with potential antioxidative activity, a group of 27 structurally diverse DHPs, as well as one pyridine compound, were studied. A group of 11 DHPs with 10-fold higher antioxidative potential than of uric acid, were further tested in cell model of human osteoblast-like cells. Short-term combined effects of DHPs and 50 µM H2O2 (1-h each), revealed better antioxidative potential of DHPs if administered before a stressor. Indirect 24-h effect of DHPs was evaluated in cells further exposed to mild oxidative stress conditions induced either by H2O2 or tert-butyl hydroperoxide (both 50 µM). Cell growth (viability and proliferation), generation of ROS and intracellular glutathione concentration were evaluated. The promotion of cell growth was highly dependent on the concentrations of DHPs used, type of stressor applied and treatment set-up. Thiocarbatone III-1, E2-134-1 III-4, Carbatone II-1, AV-153 IV-1, and Diethone I could be considered as therapeutic agents for osteoporosis although further research is needed to elucidate their bioactivity mechanisms, in particular in respect to signaling pathways involving 4-hydroxynoneal and related second messengers of free radicals.

The effects of eleven 1,4-dihydropyridine derivatives (DHPs) used alone or together with prooxidant anticancer drug doxorubicin were examined on two cancer (HOS, HeLa) and two nonmalignant cell lines (HMEC, L929). Their effects on the cell growth (3H-thymidine incorporation) were compared with their antiradical activities (DPPH assay), using well-known DHP antioxidant diludine as a reference. Thus, tested DHPs belong to three groups: (1) antioxidant diludine; (2) derivatives with pyridinium moieties at position 4 of the 1,4-DHP ring; (3) DHPs containing cationic methylene onium (pyridinium, trialkylammonium) moieties at positions 2 and 6 of the 1,4-DHP ring. Diludine and DHPs of group 3 exerted antiradical activities, unlike compounds of group 2. However, novel DHPs had cell type and concentration dependent effects on 3H-thymidine incorporation, while diludine did not. Hence, IB-32 (group 2) suppressed the growth of HOS and HeLa, enhancing growth of L929 cells, while K-2-11 (group 3) enhanced growth of every cell line tested, even in the presence of doxorubicin. Therefore, growth regulating and antiradical activity principles of novel DHPs should be further studied to find if DHPs of group 2 could selectively suppress cancer growth and if those of group 3 promote wound healing.

Although Aloe vera contains numerous bioactive components, the activity principles of widely used A. vera extracts are uncertain. Therefore, we analyzed the effects of genuine A. vera aqueous extract (AV) on human cells with respect to the effects of hydrogen peroxide (H2O2) and 4-hydroxynonenal (HNE). Fully developed A. vera leaves were harvested and analyzed for vitamin C, carotenoids, total soluble phenolic content, and antioxidant capacity. Furthermore, human cervical cancer (HeLa), human microvascular endothelial cells (HMEC), human keratinocytes (HaCat), and human osteosarcoma (HOS) cell cultures were treated with AV extract for one hour after treatment with H2O2 or HNE. The cell number and viability were determined using Trypan Blue, and endogenous reactive oxygen species (ROS) production was determined by fluorescence, while intracellular HNE–protein adducts were measured for the first time ever by genuine cell-based HNE–His ELISA. The AV extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq/g fresh weight) and cell-type-specific influence on the cytotoxicity of H2O2, as well as on endogenous production of ROS and HNE–protein adducts induced by HNE treatment, while AV itself did not induce production of ROS or HNE–protein adducts at all. This study, for the first time, revealed the importance of HNE for the activity principles of AV. Since HMEC cells were the most sensitive to AV, the effects of AV on microvascular endothelia could be of particular importance for the activity principles of Aloe vera extracts.

A recent comparison of clinical and inflammatory parameters, together with biomarkers of oxidative stress, in patients who died from aggressive COVID-19 and survivors suggested that the lipid peroxidation product 4-hydroxynonenal (4-HNE) might be detrimental in lethal SARS-CoV-2 infection. The current study further explores the involvement of inflammatory cells, systemic vascular stress, and 4-HNE in lethal COVID-19 using specific immunohistochemical analyses of the inflammatory cells within the vital organs obtained by autopsy of nine patients who died from aggressive SAR-CoV-2 infection. Besides 4-HNE, myeloperoxidase (MPO) and mitochondrial superoxide dismutase (SOD2) were analyzed alongside standard leukocyte biomarkers (CDs). All the immunohistochemical slides were simultaneously prepared for each analyzed biomarker. The results revealed abundant 4-HNE in the vital organs, but the primary origin of 4-HNE was sepsis-like vascular stress, not an oxidative burst of the inflammatory cells. In particular, inflammatory cells were often negative for 4-HNE, while blood vessels were always very strongly immunopositive, as was edematous tissue even in the absence of inflammatory cells. The most affected organs were the lungs with diffuse alveolar damage and the brain with edema and reactive astrocytes, whereas despite acute tubular necrosis, 4-HNE was not abundant in the kidneys, which had prominent SOD2. Although SOD2 in most cases gave strong immunohistochemical positivity similar to 4-HNE, unlike 4-HNE, it was always limited to the cells, as was MPO. Due to their differential expressions in blood vessels, inflammatory cells, and the kidneys, we think that SOD2 could, together with 4-HNE, be a potential link between a malfunctioning immune system, oxidative stress, and vascular stress in lethal COVID-19.

Objective Сhronic rhinosinusitis with nasal polyps (CRSwNPs) is a distinct entity within the chronic rhinosinusitis group of diseases, which are chronic upper airway diseases with several pheno- and endotypes. Oxidative stress plays an important role in the pathogenesis of CRSwNPs. The aim was to assess the association between expression of the aryl hydrocarbon receptor (AhR) and 4-hydroxynonenal (4-HNE) in patients with CRSwNPs. Methods The study included 26 patients who underwent endoscopic sinus surgery: Fourteen patients with CRSwNPs, and 12 controls with healthy sinus mucosa. Expression of AhR and 4-HNE was assessed in tissue samples using immunohistochemistry. The level of 4-HNE in serum samples was measured using the ELISA assay. Total oxidative capacity (TOC) was assessed by measuring the peroxidase activity. Results Higher levels of 4-HNE expression were observed in tissues (3, range 1–3 vs. 0, range 0–0 p<0.001) and serum (27.7±11.5 vs 9.8±7.7 pmol/mg, p < 0.001) samples of CRSwNPs patients, as compared to healthy controls. Higher expression of AhR was found in inflammatory cells (plasma cells, lymphocytes, eosinopholes) of CRSwNPs patients, compared to controls (3, range 1–3 vs. 2, range 1–2, p = 0.001). There were no differences in TOC across groups (0.0285±0.0207 vs 0.02, 978±0.0197 µM H2O2 eq., p = 0.848). Patients with bronchial asthma (57%) had abundant eosinophil in tissue samples. Patients with recalcitrant CRSwNPs had higher 4-HNE serum levels, compared to non-recalcitrant cases (27.3 vs 24.2 pmol/mg, p = 0.339). Conclusion Patients suffering from CRSwNPs have oxidative stress–mediated overexpression of AhR, which is linked to a chronic inflammatory response in the paranasal sinus tissues.

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