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On mouse neuroblastoma (Neuro-2a) and human glioblastoma (U-87 MG) cell lines, we studied the effect of inducers and inhibitors of redox-sensitive signaling system of the antioxidant-responsive element Keap1/Nrf2/ARE on the main processes that determine nerve cell viability and vital activity (proliferative activity, apoptosis, autophagy, and activation of the Keap1/Nrf2/ARE system). Inhibitors of the Keap1/Nrf2/ARE system stimulate apoptosis more pronouncedly than inducers, have a weaker effect on autophagy, and do not change the nuclear to cytoplasmic Nrf2 ratio. In general, the revealed effects testify in favor of the potential effectiveness of stimulating the Keap1/Nrf2/ARE system for the prevention and adjuvant therapy of neurodegenerative diseases., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein (RBD S-protein) contribute significantly to the humoral immune response during coronavirus infection (COVID-19) and after vaccination. The main focus of the studies of the RBD epitope composition is usually concentrated on the epitopes recognized by the virus-neutralizing antibodies. The role of antibodies that bind to RBD but do not neutralize SARS-CoV-2 remains unclear. In this study, immunochemical properties of the two mouse monoclonal antibodies (mAbs), RS17 and S11, against the RBD were examined. Both mAbs exhibited high affinity to RBD, but they did not neutralize the virus. The epitopes of these mAbs were mapped using phage display: the epitope recognized by the mAb RS17 is located at the N-terminal site of RBD (348-SVYAVNRKRIS-358); the mAb S11 epitope is inside the receptor-binding motif of RBD (452-YRLFRKSN-459). Three groups of sera were tested for presence of antibodies competing with the non-neutralizing mAbs S11 and RS17: (i) sera from the vaccinated healthy volunteers without history of COVID-19; (ii) sera from the persons who had a mild form of COVID-19; (iii) sera from the persons who had severe COVID-19. Antibodies competing with the mAb S11 were found in each group of sera with equal frequency, whereas presence of the antibodies competing with the mAb RS17 in the sera was significantly more frequent in the group of sera obtained from the patients recovered from severe COVID-19 indicating that such antibodies are associated with the severity of COVID-19. In conclusion, despite the clear significance of anti-RBD antibodies in the effective immune response against SARS-CoV-2, it is important to analyze their virus-neutralizing activity and to confirm absence of the antibody-mediated enhancement of infection by the anti-RBD antibodies.

The development of means of the prevention and treatment of age-related neurodegenerative diseases, as well as geroprotectors, among other things, is based on the inflammatory and free radical theories of aging. In this context, we studied the effect of sodium monophenol 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate (TS-13) on the behavioral and locomotor activity of C57BL/6 mice in modeling Parkinson's disease by MPTP neurotoxin injection. TS-13 administration significantly improved orientation and exploratory activity and emotional response of the animals in the open field test, but did not affect the increase in anxiety caused by MPTP injection. Long-term (6 months) TS-13 administration did not suppress spontaneous motor activity in BALB/c mice and slightly increased their exploratory activity., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3-16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

The redox-sensitive signaling system Keap1/Nrf2/ARE is a premier protective mechanism against oxidative stress that plays a key role in the pathogenesis and development of various diseases, including tuberculous granulomatous inflammation. We have previously reported that novel water-soluble phenolic antioxidant TS-13 (sodium 3-(4'-methoxyphenyl)propyl thiosulfonate) induces Keap1/Nrf2/ARE and attenuates inflammation. The aim of this study is the examination of the effect of TS-13 on tuberculous granulomatous inflammation. BALB/c mice were administered TS-13 (100 mg kg -1 day -1 ) through their drinking water starting immediately after Bacillus Calmette-Guérin (BCG) intravenous injection. Histological changes, production of reactive oxygen species (ROS) (activity of free-radical oxidation processes), and mRNA expression of Nrf2-driven, NF- κ B-, AP-1-, and autophagy-dependent signal pathway genes in the liver and peritoneal exudate were evaluated 30 days later. After the 30th day of infection, the activity of the Keap1/Nrf2/ARE system was decreased and its effector genes entailed increasing ROS production in the liver. Therapeutic intervention with TS-13 is aimed at activating the Keap1/Nrf2/ARE system that leads to an increase in Nrf2 and Nrf2-mediated gene expression and a decrease in NF- κ B expression. Changes in these pathways resulted in a decline of ROS production and a decrease in the number and the size of granulomas. In total, the results indicate that the Keap1/Nrf2/ARE system can be an effective pharmacological target in host-adjunctive treatment of tuberculosis., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2021 Elena B. Menshchikova et al.)

The free-radical theory of aging, advanced more than 50 years ago by D.Harman, remains popular today. The review analyzes age-related changes in the main endogenous mechanisms of reactive oxygen species (ROS) production and antioxidant defense mechanisms. With age, ROS generation by mitochondria, peroxisomes, and NAD(P)H oxidases is enhanced, while the transcriptional activity of the important system Keap1/Nrf2/ARE maintaining redox balance decreases. In old animals, autophagy activity is also low, which removes damaged organelles and aggregated structures from cells. The age-related shift of the redox balance towards oxidative stress can cause the development of age-associated neurodegenerative, autoimmune and inflammatory pathologies.

ROS are important intracellular messengers; their ambiguous role in malignant processes was demonstrated in many studies. The effects of a synthetic phenolic antioxidant sodium 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate sodium (TS-13) on the tumor growth and oncolytic properties of doxorubicin were studied in the experimental model of Lewis lung carcinoma in mice. In mice receiving TS-13 with drinking water (100 mg/kg), suppression of tumor growth by 32.3% was observed on day 21 after inoculation of Lewis lung carcinoma cells. Two-fold intraperitoneal injections of doxorubicin in a cumulative dose of 8 mg/kg were followed by inhibition of tumor growth by 49.5%. Combined treatment with TS-13 and doxorubicin suppressed the tumor growth by 55.4%. In contrast to doxorubicin, TS-13 inhibited NO generation by peritoneal macrophages. The results show the prospect of studying TS-13 in the context of overcoming drug-resistance of tumors.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

We studied differences in the production of pro- and anti-inflammatory cytokines and IRF3 transcription factor by peritoneal macrophages from mice of opposite strains CBA/J and C57Bl/6 and the effect of 60-kDa oxidized dextran on these parameters. Macrophages from C57Bl/6 mice were mainly characterized by the production of proinflammatory cytokines TNFα, IL-12, and MCP-1 (markers of M1 polarization). By contrast, CBA/J mice exhibited a relatively high level of anti-inflammatory cytokine IL-10 and lower expression of proinflammatory cytokines (M2 phenotype). IRF3 content in peritoneal macrophages of CBA/J mice was higher than in C57Bl/6 mice. Oxidized dextran decreased the expression of IRF3 upon stimulation of cells from CBA/J mice with LPS, but increased this process in C57Bl/6 mice. Despite a diversity of oxidized dextran-induced changes in cytokine production, the data confirm our hypothesis that this agent can stimulate the alternative activation of macrophages.

Octahedral rhenium cluster complexes have recently emerged as relevant building blocks for the design of singlet oxygen photosensitizing materials toward biological applications such as blue-light photodynamic therapy. However, their singlet oxygen generation ability as well as biological properties have been studied only superficially. Herein we investigate in detail the singlet oxygen photogeneration, dark and photoinduced cytotoxicity, cellular uptake kinetics, cellular localization and in vitro photoinduced oxidative stress, and photodynamic cytotoxicity of the series of octahedral rhenium cluster complexes [{Re 6 Q 8 }(CN) 6 ] 4- , where Q = S, Se, Te. Our results demonstrate that the selenium-containing complex possesses optimal properties in terms of absorption and singlet oxygen productivity. These features coupled with the cellular internalization and low dark toxicity lead to the first photoinduced cytotoxic effect observed for a molecular [{M 6 Q 8 }L 6 ] complex, making it a promising object for further study in terms of blue-light PDT.

Influenza virus hemagglutinins are surface proteins responsible for fusion of the viral and cellular membranes. Their capacity to mediate membrane fusion (fusogenic activity) is studied by various methods, including the syncytium formation and pseudovirus transduction methods. We constructed plasmids coding for genes of three H1 and one H5 hemagglutinins and compared their fusogenic activities. Hemagglutinin capacity to induce syncytium formation did not always correlate with the transduction activity of the respective pseudoviruses. Hemagglutinin H5 exhibited high fusogenic activity in studies by both methods, however, two of the studied H1 hemagglutinins induced the formation of syncytia, but did not mediate pseudovirus transduction. This could be due to different capsid sizes of influenza virus and vesicular stomatitis virus, which determines their different permeability through the fusion pore.

Nrf2 transcription factor plays a key role in maintaining cellular redox balance under stress and is a perspective target for oxidative stress-associated diseases. Under normal conditions, Nrf2 transcriptional activity is low due to its rapid ubiquitination and degradation in the 26S proteasome, as well as through various modifications of amino acid residues of this transcription factor that regulate its transport to the nucleus and binding to DNA. Continuous activation of Nrf2 is possible due to autophagy and epigenetic regulation that may underlie the increased resistance of tumor cells to radiotherapy and chemotherapy. This review deals with the mechanisms of regulation of Nrf2 transcriptional activity and its main elements, and pharmacological approaches to activation of the Keap1/Nrf2/ARE system.

Many plant phenols (stilbenes, curcumins, catechins, flavonoids, etc.) are effective antioxidants and protect cells during oxidative stress. Extensive clinical studies on the potential of phenolic compounds for treatment of cardiovascular, neurodegenerative, oncological, and inflammatory diseases are now being conducted. In addition to direct antioxidant effect, plant phenols may provide a protective effect via activation of the Keap1/Nrf2/ARE redox-sensitive signaling system and regulation of autophagy. In this review, mechanisms of effects of the most common plant phenols on autophagy are presented.

Differences in peritoneal macrophage polarization in mice of opposite lines CBA and C57Bl/6 and the effects of 60 kDa oxidized dextran were studied. Macrophages of C57Bl/6 mice demonstrated a phenotype close to M1, with increasing expression of CD86 costimulatory molecule and unchanged CD206 expression in response to activation. Macrophages of CBA mice demonstrated higher plasticity in response to activating agents; expression of the markers increased irrespectively on stimulated receptor (TLR-4 or mannose receptor) and both CD86 (classical activation) and CD206 (alternative activation) increased. Macrophage response to addition of oxidized dextran (60 kDa) to the culture medium could be characterized as potentiation of their alternative activation: expression of CD86 in CBA mice in response to LPS and LPS+IL-4 and in C57Bl/6 mice in response to IFN-γ and LPS+IFN-γ decreased, while expression of CD206 by intact macrophages of CBA mice and by macrophages stimulated by IFN-γ and IL-4 increased under the effect of 60 kDa oxidized dextran.

We studied the effects of liposomal pharmaceutical compositions with oxidized dextrans on functional activity of U937 monocyte/macrophage-like cells. Liposomes in the emulsion contained oxidized dextran with a molecular weights of 40 kDa or 70 kDa or isonicotinic acid hydrazide (INAH) conjugated with oxidized dextran (40 kDa). Cell viability was evaluated by MTT test; mitochondrial transmembrane potential and production of superoxide anion and H2O2 were studied by fluorescent methods. The studied compositions exhibited no cytotoxic effect and even improved cell viability and mitochondrial respiration. Liposomes with oxidized 40 kDa dextran, including those with INAH-conjugated dextran, inhibited production of superoxide anion, but increased H2O2 generation.

We studied ROS production by HaCaT keratinocytes, the state of transmembrane mitochondrial potential, and activation of transcription factor Nrf2 in response to brine exposure. It was demonstrated that this exposure induces rapid but moderate decrease in mitochondrial potential, stimulates ROS production, and leads to activation of transcription factor Nrf2.

We studied peculiarities of morphofunctional organization of the immune system in C57Bl/6g and CBA mice differing by their susceptibility to various types of infectious agents. The revealed differences in the structure of lymphoid organs, T lymphocyte subpopulation ratio and their differentiation into Th1/Th2 cells after mitogen stimulation drove us to a conclusion on genetically determined regularities in the development of the immune response in these animal strains.