BackgroundPulmonary fibrosis (PF) is a severe disease leading to disability and death. It is important to search for novel therapeutic agents, for which a model of bleomycin-induced PF is used and pirfenidone and dexamethasone are used as comparators. The aim of the study was to establish the effect of comparators on model PF.MethodsWe induced PF in male SD rats by intratracheal administration of 2.5 mg/kg bleomycin (BLM). Dexamethasone was administered intravenously (0.5 mg/kg, 7 days,), and pirfenidone was administered orally (50 mg/kg, 21 day). Clinical signs of health deviations, body weight, weight gain, food consumption and spirometry were monitored. The lung condition, cellular composition of the bronchoalveolar fluid (BALF), the lung concentration of hydroxyproline, blood biochemistry and the severity of fibrosis were analyzed 8 and 22 days post PF induction.ResultsAll animals administered BLM intratracheally developed pronounced signs of PF, including weight loss leading to pulmonary cachexia, deterioration in general condition, increased respiratory rate and decreased vital capacity, increased hydroxyproline levels in lung tissue, elevated level of neutrophils, lymphocytes, and pulmonary macrophages in the BALF. All of these features developed over time and were evident on day 8 and 22 of the study. Histological analysis revealed a pronounced inflammatory response on day 8 and a generalized fibrotic process on day 22. Dexamethasone and pirfenidone not only failed to improve clinical condition of animals, but even worsened it; one animal receiving pirfenidone died.ConclusionAnimals with PF treated with dexamethasone and pirfenidone showed no improvement compared to untreated animals. Although dexamethasone reduced hydroxyproline levels, this did not improve either the animals’ general condition or lung damage.

Most intracellular proteins are degraded by the ubiquitin-proteasome system (UPS), with proteasomes directly hydrolyzing protein substrates. Specific forms of proteasomes (non-constitutive proteasomes), implicated in antigen presentation, cellular homeostasis maintenance and stress response have been described. However, proteasomes were also identified outside cells, where their function remains unclear. Proteasome secretion via extracellular vesicles (EVs) have been reported, though the direct transmission of non-constitutive proteasomes between cells has not been shown. Using genetically modified cells, including a human adenocarcinoma cell line SW620B8-mCherry expressing the β5i subunit of non-constitutive proteasomes fused to the mCherry protein, and a number of techniques, such as differential centrifugation, affinity isolation, unspecific precipitation, NTA and microscopy, EVs containing non-constitutive proteasomes were obtained and characterized. Different cell lines were shown to secrete varying amounts of vesicles containing non-constitutive proteasomes. The content of these proteasomes in EVs was increased after the stimulation of cells with IFN-γ. The interaction of vesicles secreted by SW620B8-mCherry cells with recipient cells was demonstrated. The β5i-mCherry chimera was detected in lysates of different recipient cells following incubation with EVs secreted from SW620B8-mCherry cells. The obtained results highlight the transfer of non-constitutive proteasomes from one cell to another via EVs.

Virus-neutralizing peptides (VNPs) emerged as promising antiviral drug candidates with unprecedented specificity and cost-effectiveness during the recent COVID-19 pandemic. However, limited avidity, lack of effector functions, short circulatory half-life, and restricted administration routes make them inferior compared to neutralizing antibodies. To address these constraints, a potent VNP that targets the SARS-CoV-2 S protein is combined with Barnase, a highly active RNA-cleaving enzyme from Bacillus amyloliquefaciens. The resulting LCB1-Barnase (LCB1-Bn) chimera retains strong binding affinity for the SARS-CoV-2 S protein and demonstrates a fourfold reduction in IC50 compared to the LCB1 peptide alone in competitive ELISA and in in vitro neutralization tests. In transgenic CAG-hACE2 mice infected with wild-type SARS-CoV-2, intranasal administration of LCB1-Bn significantly improves survival and reduces viral load by 29-fold. To extend circulation life and allow systemic intravenous administration, an albumin-binding domain (ABD) from Streptococcus protein G is added to LCB1-Bn, producing LCB1-ABD-Bn fusion protein which displays a 95-fold increase in serum half-life. LCB1-ABD-Bn exhibits good tolerability at doses below 10 mg/kg and provides protection of SARS-CoV-2-infected CAG-hACE2 animals in 24-hour post-infection intraperitoneal treatment. Cryo-EM reveals the LCB1-ABD-Bn’s tight interaction with S protein RBD domains, highlighting its potential as a promising drug candidate against SARS-CoV-2.

Proteasomes play a central role in antiviral and antitumor cytotoxic immunity, generating most of the >10,000 unique proteome-derived peptides bound to ∼200,000 human leukocyte antigen (HLA) class I molecules on each human cell. Here, we present a protocol for hybrid mass spectrometry-AI mapping of proteasome-generated HLA class I epitopes. We describe steps for in vitro processing of antigens by proteasomes, HLA class I allele selection, and peptide pool preparation. We then detail procedures for determining HLA class I ligands and population immunity. For complete details on the use and execution of this protocol, please refer to Kudriaeva et al.1.

The COVID-19 pandemic was the most dramatic in the newest history with nearly 7 million deaths and global impact on mankind. Here, we report binding index of 305 human leukocyte antigen (HLA) class I molecules from 18,771 unique haplotypes of 28,104 individuals to 821 peptides experimentally observed from spike protein receptor binding domain (RBD) of five main SARS-CoV-2 strains hydrolyzed by human proteasomes with constitutive and immune catalytic phenotypes. Our data read that mutations in the human angiotensin-converting enzyme 2 (hACE2)-binding region RBD496-513 of Omicron B.1.1.529 strain results in a dramatic increase of proteasome-mediated release of two public HLA class I epitopes. Global population analysis of HLA class I haplotypes, specific to these peptides, demonstrated decreased mortality of human populations enriched in these haplotypes from COVID-19 after but not before December, 2021, when Omicron became dominant SARS-CoV-2 strain. Noteworthy, currently circulating BA.2.86 and JN.1 strains contain same amino acid substitutions at key proteasomal cleavage sites, thus preserving identified core epitopes.

Spinal cord injury (SCI) is a medical challenge that results in the formation of a glial scar preventing recovery of axonal conductivity. Cytokines and chemokines significantly affect the pathogenesis of SCI and represent important targets for therapeutic intervention. Here, dozens of cytokines and chemokines are dynamically monitored in plasma, cerebrospinal fluid, and injury site released in response to experimental SCI conducted in two rodent strains and patients undergoing surgical removal of intramedullary tumors. Dataset comprising 6,172 cytokine/chemokine values across 8 time points suggests that SCI in mammals is accompanied by a massive cytokine storm in cerebrospinal fluid, mainly driven by CXCL1, IL-6, and CCL2-5. Sub-acute phase is mostly associated with IL-2, IL-7, CCL22 and CX3CL1, whereas TNFα and IL17α permanently persists in CNS for even weeks following SCI. The effects of mogamulizumab and chemical antagonists of C-C/C-X-C chemokine receptors TAK-799, SB225002, and MK-7123 on SCI recovery in rodents are further estimated. Here blockade of CCR5 and CXCR1/2 chemokine receptors is shown beneficial for amelioration of acute SCI, whereas anti-CCR4 antibody mogamulizumab readily prevents secondary inflammation in the injured area. Summarizing, the current report claims for a novel combined time-resolved therapeutic modality in SCI treatment, which supports feasibility and motivates off-label clinical evaluation in appropriate cohorts.

Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.

Analysis of the mechanisms underlying the occurrence and progression of cancer represents a key objective in contemporary clinical bioinformatics and molecular biology. Utilizing omics data, particularly transcriptomes, enables a detailed characterization of expression patterns and post-transcriptional regulation across various RNA types relative to the entire transcriptome. Here, we assembled a dataset comprising transcriptomic data from approximately 16 000 patients encompassing over 160 types of cancer. We employed state-of-the-art gradient boosting algorithms to discern intricate correlations in the expression levels of four clinically significant microRNAs, specifically, hsa-mir-21, hsa-let-7a-1, hsa-let-7b, and hsa-let-7i, with the expression levels of the remaining 60 660 unique RNAs. Our analysis revealed a dependence of the expression levels of the studied microRNAs on the concentrations of several small nucleolar RNAs and regulatory long noncoding RNAs. Notably, the roles of these RNAs in the development of specific cancer types had been previously established through experimental evidence. Subsequent evaluation of the created database will facilitate the identification of a broader spectrum of overarching dependencies related to changes in the expression levels of various RNA classes in diverse cancers. In future, it will make possible to discover unique alterations specific to certain types of malignant transformations.

In this study, we evaluated the ability of the synthetic amphipathic helical peptide (SAHP), L-37pA, which mediates pathogen recognition and innate immune responses, to treat acute respiratory distress syndrome (ARDS) accompanied by diffuse alveolar damage (DAD) and chronic pulmonary fibrosis (PF). For the modeling of ARDS/DAD, male ICR mice were used. Intrabronchial instillation (IB) of 200 µL of inflammatory agents was performed by an intravenous catheter 20 G into the left lung lobe only, leaving the right lobe unaffected. Intravenous injections (IVs) of L-37pA, dexamethasone (DEX) and physiological saline (saline) were used as therapies for ARDS/DAD. L37pA inhibited the circulating levels of inflammatory cytokines, such as IL-8, TNFα, IL1α, IL4, IL5, IL6, IL9 and IL10, by 75–95%. In all cases, the computed tomography (CT) data indicate that L-37pA reduced lung density faster to −335 ± 23 Hounsfield units (HU) on day 7 than with DEX and saline, to −105 ± 29 HU and −23 ± 11 HU, respectively. The results of functional tests showed that L-37pA treatment 6 h after ARDS/DAD initiation resulted in a more rapid improvement in the physiological respiratory lung by 30–45% functions compared with the comparison drugs. Our data suggest that synthetic amphipathic helical peptide L-37pA blocked a cytokine storm, inhibited acute and chronic pulmonary inflammation, prevented fibrosis development and improved physiological respiratory lung function in the ARDS/DAD mouse model. We concluded that a therapeutic strategy using SAHPs targeting SR-B receptors is a potential novel effective treatment for inflammation-induced ARDS, DAD and lung fibrosis of various etiologies.

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome–lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS.

Rheumatoid arthritis(RA)is an autoimmune disorder char-acterized by synovial joint damage and progressive loss of mobility.The human leukocyte antigen(HLA)class Ⅱ alleles HLA-DRB1*01:01 and HLA-DRB1*04:01 are strongly linked to RA susceptibility.Several autoantigenic peptides were re-ported to bind to RA-associated HLA-Ⅱ and trigger autor-eactive CD4+T cell response.Here,we propose a dual combinatorial approach to identify novel autoantigenic peptides presented by HLA-Ⅱ.

Ubiquitination is an evolutionary, ancient system of post-translational modification of proteins that occurs through a cascade involving ubiquitin activation, transfer, and conjugation. The maturation of this system has followed two main pathways. The first is the conservation of a universal structural fold of ubiquitin and ubiquitin-like proteins, which are present in both Archaea and Bacteria, as well as in multicellular Eukaryotes. The second is the rise of the complexity of the superfamily of ligases, which conjugate ubiquitin-like proteins to substrates, in terms of an increase in the number of enzyme variants, greater variation in structural organization, and the diversification of their catalytic domains. Here, we examine the diversity of the ubiquitination system among different organisms, assessing the variety and conservation of the key domains of the ubiquitination enzymes and ubiquitin itself. Our data show that E2 ubiquitin-conjugating enzymes of metazoan phyla are highly conservative, whereas the homology of E3 ubiquitin ligases with human orthologues gradually decreases depending on "molecular clock" timing and evolutionary distance. Surprisingly, Chordata and Echinodermata, which diverged over 0.5 billion years ago during the Cambrian explosion, share almost the same homology with humans in the amino acid sequences of E3 ligases but not in their adaptor proteins. These observations may suggest that, firstly, the E2 superfamily already existed in its current form in the last common metazoan ancestor and was generally not affected by purifying selection in metazoans. Secondly, it may indicate convergent evolution of the ubiquitination system and highlight E3 adaptor proteins as the "upper deck" of the ubiquitination system, which plays a crucial role in chordate evolution.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

Lymphotoxin α and lymphotoxin β (LTs), TNF superfamily members, are expressed in either soluble (LTα 3 ) or membrane-bound (LTα 1 β 2 or LTα 2 β 1 ) forms. In the pathological context, LT-mediated signaling is known to exacerbate autoimmunity by perpetuating inflammation and promoting the formation of tertiary lymphoid organs. Despite this understanding, the exact roles of LTα and LTβ in the pathogenesis of the murine model of multiple sclerosis, and experimental autoimmune encephalomyelitis (EAE), remain controversial. Here, we employed a panel of gene-modified mice with cell-type restricted ablation of LTα (targeting both membrane-bound and soluble forms of LTs) to unravel the contributions of LTs from various lymphoid cells, namely T cells, type 3 innate lymphoid cells (ILC3) and B cells, in EAE. We found that the effects of LTα deletion were dependent on the cellular source. ILC3-derived lymphotoxins exerted a protective role in EAE by regulating the accumulation of IFN-ɣ- and GM-CSF-producing T H cells in the CNS. In contrast, T-cell-derived lymphotoxins promoted IL-17A- and GM-CSF-mediated T H responses in the periphery, whereas B-cell-derived lymphotoxins were pathogenic only in the autoantibody-mediated EAE model. Collectively, our findings unveil the multifaceted involvement of lymphotoxins in EAE pathogenesis and challenge the view that lymphotoxins play a solely pathogenic role in neuroinflammation.

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide–MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols.

Ubiquitination, one of the most common posttranslational modifications of proteins, has a significant impact on their functions, such as stability, activity, and cellular localization. Disorders in the ubiquitination and deubiquitination processes are associated with various oncological and neurodegenerative diseases. The complexity of ubiquitin signaling, specifically monoubiquitination and polyubiquitination with different lengths and types of ubiquitin– ubiquitin linkages determines their versatility and ability to regulate hundreds of different cellular processes. Advanced biochemical, mass spectrometric, and computational studies are required for in-depth understanding of the mechanisms of assembly and disassembly, as well as detection of ubiquitin chains and their signal transmission. Recent scientific achievements make it possible to identify protein ubiquitination and the structure of ubiquitin chains, but there are a lot of issues in this area to be clarified. The present review provides a detailed analysis of the current understanding of the architectonics of ubiquitin chains.

Background: The genotypic variability of the SARS-CoV-2 virus has proven to be extremely high, and the emergence of new strains raises concerns about their possible high virulence, transmissibility, and ability to bypass responses of the body's immune system induced by previous infection or vaccination. Therefore, one of the main tasks is to study the pathogenesis of various variants of the virus using experimental animal biomodels of SARS-CoV-2 to quickly find methods and approaches to fighting new viruses. Methods: 60 humanized mice of the C57BL/6-Tgtn (CAG-human AEC2-IRES-Luciferase-WPRE-polyA) line (hACE2) were used. Mice were infected intranasally at different doses with three variants of the SARS-CoV-2 virus: Wuhan, Delta and Omicron. Results: We showed that humanized hACE2 mice, when infected with all three variants of the SARS-CoV-2 virus, showed typical pathological changes in lung consistency comparable to those found in COVID-19 in humans. All mice developed interstitial pneumonia, characterized by inflammatory cell infiltration and thickening of the alveolar septa, characteristic of vascular damage. Conclusions: At a dose of 4 lg plaque-forming unit (PFU), all variants showed 100% mortality. A dose-dependent effect was established only for the Wuhan and Delta variants. In a comparative assessment of different variants of the SARS-CoV-2 virus in a humanized mouse model of hACE2, it was found that the Delta variant leads to more severe damage compared to Wuhan or Omicron. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: The acute respiratory distress syndrome (ARDS), secondary to viral pneumonitis, is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019)—ongoing SARS-CoV-2 infection— reached more than 0.7 billion registered cases.Methods: Recently, we elaborated a non-surgical and reproducible method of the unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice–a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data read that two C–C chemokine receptor 5 (CCR5) ligands, macrophage inflammatory proteins (MIPs) MIP-1α/CCL3 and MIP-1β/CCL4, are upregulated in this DAD model up to three orders of magnitude compared to the background level.Results: Here, we showed that a nonpeptide compound TAK-779, an antagonist of CCR5/CXCR3, readily prevents DAD in the lung with a single injection of 2.5 mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung and mononuclear infiltration of the wall and lumen of the alveoli in the TAK-779-treated animals. Administration of TAK-779 decreased the 3–5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1α/β, MCP-1, and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779-treated animals.Discussion: Our pre-clinical data suggest that TAK-779 is more effective than the administration of dexamethasone or the anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors for the treatment of virus-induced hyperinflammation syndromes, including COVID-19.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

Antigen presentation by major histocompatibility complex class II (MHC-II) molecules is crucial for eliciting an efficient immune response by CD4+ T cells and maintaining self-antigen tolerance. Some MHC-II alleles are known to be positively or negatively associated with the risk of the development of different autoimmune diseases (ADs), including those characterized by the emergence of autoreactive T cells. Apparently, the MHC-II presentation of self-antigens contributes to the autoimmune T cell response, initiated through a breakdown of central tolerance to self-antigens in the thymus. The appearance of autoreactive T cell might be the result of (i) the unusual interaction between T cell receptors (TCRs) and self-antigens presented on MHC-II; (ii) the posttranslational modifications (PTMs) of self-antigens; (iii) direct loading of the self-antigen to classical MHC-II without additional nonclassical MHC assistance; (iv) the proinflammatory environment effect on MHC-II expression and antigen presentation; and (v) molecular mimicry between foreign and self-antigens. The peculiarities of the processes involved in the MHC-II-mediated presentation may have crucial importance in the elucidation of the mechanisms of triggering and developing ADs as well as for clarification on the protective effect of MHC-II alleles that are negatively associated with ADs.