Search

Aim.Thе purpose of the work was to develop a new construction of enzyme biosensor based on creatinine deiminase for highly sensitive creatinine determination. Methods. A new construction of enzyme biosensor based on creatinine deiminase was developed for the creatinine determination. A differential pair of gold interdigitated electrodes deposited onto a ceramic substrate was used as the electrochemical transducer. Creatinine deiminase was immobilized by cross-linking with glutaraldehyde on the surface of electrodes. Results. The biosensor showed high sensitivity towards creatinine, the limit of detection was 5 µM. The biosensor was characterized by wide linear range of creatinine determination, high reproducibility of responses and showed high storage stability – after 50 days storage the biosensor retained 83% of the initial response value. Conclusions. In future the developed biosensor can be used for express evaluation of the creatinine in biological samples.

Treatment with immune checkpoint inhibitors (ICIs) has improved the prognosis of melanoma patients. However, ICIs can cause an overactivation of the immune system followed by diverse immunological side effects known as immune-related adverse events (irAE). Currently, the toxicity of irAE is limiting the usage of ICIs. Here, we studied circulating monocytic myeloid-derived suppressor cells (M-MDSCs) and T cells in course of irAE after the ICI therapy. Our longitudinal study involved 31 melanoma patients with and without adverse events during anti-PD-1 monotherapy or anti-CTLA-4/PD-1 combination therapy. Peripheral blood samples were analyzed before ICI start, during ICI treatment, at the time point of irAE and during immunosuppressive treatment to cure irAE. We observed an enhanced progression-free survival among patients with irAE. In patients with irAE, we found an upregulation of CD69 on CD8+ T cells and a decreased frequency of regulatory T cells (Tregs). Moreover, lower frequencies of Tregs correlated with more severe side effects. Patients treated with immunomodulatory drugs after irAE manifestation tend to show an elevated number of M-MDSCs during an immunosuppressive therapy. We suggest that an activation of CD8+ T cells and the reduction of Treg frequencies could be responsible for the development of irAE.

The article deals with the problem of using information technologies in the process of specialized training of students of engineering and pedagogical specialties. The aim of the article is to reveal the essence and peculiarities of the use of information technology in the training of future vocational teachers; to identify the problems of classical teaching methods and to find ways to solve them through the use of information technology. A set of research methods was used, namely: content analysis, generalization and specification of the provisions of the Standards of Higher and Vocational Education of Ukraine, educational programs and curricula, designing the content and structure of an e-learning course, substantiation of the conditions for using e-learning courses in the process of specialized training of students. Results of the study. The Standard of Higher Education of Ukraine, specialty 015 Vocational Education (by specialization), first (bachelor) level, was analyzed. The existing curricula for the training of vocational teachers of transport profile were examined for their compliance with the requirements of educational standards. It has been determined that the normative part of the curriculum includes cycles of general and professional training which contain blocks of fundamental, psychological, pedagogical and methodological, scientific and subject, and practical training. It has been found out that the courses that form the special competencies of vocational teachers of transport profile belong to the block of scientific and subject training. The courses that form the special competencies of transport vocational teachers are highlighted, namely: vehicles and their maintenance, transport logistics, electrical and information-computer systems in transport, traffic rules, operation and repair of transport machine engines, interchangeability, standardization and technical measurements, and technical mechanics. Traditional approaches to the study of these courses are investigated in order to identify the main shortcomings. It has been stated that the traditional methodology of conducting classes involves significant time spent on note-taking of educational material, which can be mastered with greater efficiency using information and communication tools. Ways to eliminate the shortcomings of traditional approaches to the study of specialized courses are proposed, one of which is the development and implementation of e-courses on the Moodle platform, since this platform allows combining different formats of information, makes it possible to timely update the educational material in accordance with the pace of development of the transport industry, as well as it is easy to use and supported by most modern Smart devices. The structural and functional components of the e-learning course are defined including an information and organizational component; a training component; an information and reference component; and a practical component. The requirements for creating and implementing e-learning courses in the educational process are generalized. An e-learning course on the Moodle platform in the Transport Logistics course has been developed. It consists of information and methodological, training, control and evaluation, and reference blocks. A description of the content of these structural blocks is presented. Prospects for further research are identified; these are development of methods for using e-learning courses in specialized subjects in a mixed form of education under martial law.

Despite the remarkable success of immune checkpoint inhibitors (ICIs) in melanoma treatment, resistance to them remains a substantial clinical challenge. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells that can suppress antitumor immune responses mediated by T and natural killer cells and promote tumor growth. They are major contributors to ICI resistance and play a crucial role in creating an immunosuppressive tumor microenvironment. Therefore, targeting MDSCs is considered a promising strategy to improve the therapeutic efficacy of ICIs. This Review describes the mechanism of MDSC-mediated immune suppression, preclinical and clinical studies on MDSC targeting, and potential strategies for inhibiting MDSC functions to improve melanoma immunotherapy.

PurposeAlthough the treatment of advanced melanoma patients with immune checkpoint inhibitors (ICI) significantly increased the therapeutic efficiency, many patients remain resistant to ICI that could be due to immunosuppression mediated by myeloid-derived suppressor cells (MDSC). These cells are enriched and activated in melanoma patients and could be considered as therapeutic targets. Here we studied dynamic changes in immunosuppressive pattern and activity of circulating MDSC from melanoma patients treated with ICI.Experimental designMDSC frequency, immunosuppressive markers and function were evaluated in freshly isolated peripheral blood mononuclear cells (PBMC) from 29 melanoma patients receiving ICI. Blood samples were taken prior and during the treatment and analyzed by flow cytometry and bio-plex assay.ResultsMDSC frequency was significantly increased before the therapy and through three months of treatment in non-responders as compared to responders. Prior to the ICI therapy, MDSC from non-responders displayed high levels of immunosuppression measured by the inhibition of T cell proliferation assay, whereas MDSC from responding patients failed to inhibit T cells. Patients without visible metastasis were characterized by the absence of MDSC immunosuppressive activity during the ICI treatment. Moreover, non-responders showed significantly higher IL-6 and IL-8 concentrations before therapy and after the first ICI application as compared to responders.ConclusionsOur findings highlight the role of MDSC during melanoma progression and suggest that frequency and immunosuppressive activity of circulating MDSC before and during the ICI treatment of melanoma patients could be used as biomarkers of response to ICI therapy.

Introduction Melanoma accounts for the vast majority of skin cancer-related deaths. It is known for its high immunogenicity, which makes the disease a suitable target for immunotherapies (1) that deploy [...], Despite the remarkable success of immune checkpoint inhibitors (ICIs) in melanoma treatment, resistance to them remains a substantial clinical challenge. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells that can suppress antitumor immune responses mediated by T and natural killer cells and promote tumor growth. They are major contributors to ICI resistance and play a crucial role in creating an immunosuppressive tumor microenvironment. Therefore, targeting MDSCs is considered a promising strategy to improve the therapeutic efficacy of ICIs. This Review describes the mechanism of MDSC-mediated immune suppression, preclinical and clinical studies on MDSC targeting, and potential strategies for inhibiting MDSC functions to improve melanoma immunotherapy.

A gradual decay in humoral and cellular immune responses over time upon SAR1S-CoV-2 vaccination may cause a lack of protective immunity. We conducted a longitudinal analysis of antibodies, T cells, and monocytes in 25 participants vaccinated with mRNA or ChAdOx1-S up to 12 weeks after the 3rd (booster) dose with mRNA vaccine. We observed a substantial increase in antibodies and CD8 T cells specific for the spike protein of SARS-CoV-2 after vaccination. Moreover, vaccination induced activated T cells expressing CD69, CD137 and producing IFN-γ and TNF-α. Virus-specific CD8 T cells showed predominantly memory phenotype. Although the level of antibodies and frequency of virus-specific T cells reduced 4-6 months after the 2nd dose, they were augmented after the 3rd dose followed by a decrease later. Importantly, T cells generated after the 3rd vaccination were also reactive against Omicron variant, indicated by a similar level of IFN-γ production after stimulation with Omicron peptides. Breakthrough infection in participants vaccinated with two doses induced more SARS-CoV-2-specific T cells than the booster vaccination. We found an upregulation of PD-L1 expression on monocytes but no accumulation of myeloid cells with MDSC-like immunosuppressive phenotype after the vaccination. Our results indicate that the 3rd vaccination fosters antibody and T cell immune response independently from vaccine type used for the first two injections. However, such immune response is attenuated over time, suggesting thereby the need for further vaccinations.

Background Tumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors. Their interactions with myeloid cells lead to the generation of myeloid-derived suppressor cells (MDSC), which inhibit the antitumor function of T and NK cells. We demonstrated previously that EV derived from mouse and human melanoma cells induced immunosuppressive activity via increased expression of programmed cell death ligand 1 (PD-L1) on myeloid cells that was dependent on the heat-shock protein 90α (HSP90α) in EV. Here, we investigated whether soluble HSP90α could convert monocytes into MDSC.Methods CD14 monocytes were isolated from the peripheral blood of healthy donors, incubated with human recombinant HSP90α (rHSP90α) alone or in the presence of inhibitors of TLR4 signaling and analyzed by flow cytometry. Inhibition of T cell proliferation assay was applied to assess the immunosuppressive function of rHSP90α-treated monocytes. HSP90α levels were measured by ELISA in plasma of patients with advanced melanoma and correlated with clinical outcome.Results We found that the incubation of monocytes with rHSP90α resulted in a strong upregulation of PD-L1 expression, whereas reactive oxygen species (ROS) and nitric oxide (NO) production as well as the expression of arginase-1, ectoenzymes CD39 and CD73 remained unchanged. The PD-L1 upregulation was blocked by anti-TLR4 antibodies and a nuclear factor-κB inhibitor. rHSP90α-treated monocytes displayed the downregulation of HLA-DR expression and acquired the resistance to apoptosis. Moreover, these monocytes were converted into MDSC as indicated by their capacity to inhibit T cell proliferation, which was mediated by TLR4 signaling as well as PD-L1 and indoleamine 2,3-dioxygenase (IDO) 1 expression. Higher levels of HSP90α in plasma of patients with melanoma correlated with augmented PD-L1 expression on circulating monocytic (M)-MDSC. Patients with melanoma with high levels of HSP90α displayed shorter progression-free survival (PFS) on the treatment with immune checkpoint inhibitors (ICIs).Conclusion Our findings demonstrated that soluble rHSP90α increased the resistance of normal human monocytes to apoptosis and converted them into immunosuppressive MDSC via TLR4 signaling that stimulated PD-L1 and IDO-1 expression. Furthermore, patients with melanoma with high concentrations of HSP90α displayed increased PD-L1 expression on M-MDSC and reduced PFS after ICI therapy, suggesting HSP90α as a promising therapeutic target for overcoming immunosuppression in melanoma.

The number and stability of lysosomes (LYs) are different in cancer and healthy cells that makes them a possible target for cancer specific therapy. However, no LY‐targeting drug is clinically approved yet. We describe in this paper a new therapeutic approach based on alkylation of lysosomal thiols in cancer cells by reversible thiol binder 11. The treatment with 11 increases the level of lysosomal reactive oxygen species leading to their destabilization, disruption and immunogenic cancer cell death. These effects are not observed in healthy cells. In murine sarcoma Nemeth‐Kellner (NK)/Ly‐RB model, 11 exhibits the spectacular therapeutic effect: it extends the lifespan of the treated mice from 21 to 85 days and cures 40 % of mice. The survived mice develop antibodies against tumor NK/Ly‐RB cells. Their repeated challenge with the NK/Ly‐RB cells results in 100 % mice survival compared to 0 % survival in the control group of naïve mice. Ex vivo data indicate that neutrophils in spleen of the cured animals are also involved in targeting cancer cells and produce neutrophil extracellular traps. In summary, 11 induces the direct antitumor effect supported by humoral immune responses, as well as priming neutrophil's reaction against tumors. [ABSTRACT FROM AUTHOR]

Nanoformulations for delivering nucleotides into cells as vaccinations as well as treatment of various diseases have recently gained great attention. Applying such formulations for a local treatment strategy, e.g., for cancer therapy, is still a challenge, for which improved delivery concepts are needed. Hence, this work focuses on the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) for a prospective “magnetofection” application. By functionalizing SPIONs with an active catechol ester (CafPFP), polyethyleneimine (PEI) was covalently bound to their surface while preserving the desired nanosized particle properties with a hydrodynamic size of 86 nm. When complexed with plasmid-DNA (pDNA) up to a weight ratio of 2.5% pDNA/Fe, no significant changes in particle properties were observed, while 95% of the added pDNA was strongly bound to the SPION surface. The transfection in A375-M cells for 48 h with low amounts (10 ng) of pDNA, which carried a green fluorescent protein (GFP) sequence, resulted in a transfection efficiency of 3.5%. This value was found to be almost 3× higher compared to Lipofectamine (1.2%) for such low pDNA amounts. The pDNA-SPION system did not show cytotoxic effects on cells for the tested particle concentrations and incubation times. Through the possibility of additional covalent functionalization of the SPION surface as well as the PEI layer, Caf-PEI-SPIONs might be a promising candidate as a magnetofection agent in future.

The electrochemical pulsewise method for corrosion and mechanical damage monitoring the process equipment operated in liquid electrolyte agent is provided. The developed method makes it possible to inspect the equipment condition either according to the formation rate of disturb pulses which are recorded during a certain period of time or according to the equipment potential behave our in time.

Despite their long history and their synthetic potential underlined by various recent advances, radical thiol‐yne coupling reactions have so far only rarely been exploited for the functionalization of biomolecules, and no examples yet exist for their application in live cells ‐ although natural thiols show widespread occurrence therein. By taking advantage of the particular cellular conditions of mitochondria in cancer cells, we have demonstrated that radical thiol‐yne coupling represents a powerful reaction principle for the selective targeting of these organelles. Within our studies, fluorescently labeled reactive alkyne probes were investigated, for which the fluorescent moiety was chosen to enable both mitochondria accumulation as well as highly sensitive detection. After preliminary studies under cell‐free conditions, the most promising alkyne‐dye conjugates were evaluated in various cellular experiments comprising analysis by flow cytometry and microscopy. All in all, these results pave the way for improved future therapeutic strategies relying on live‐cell compatibility and selectivity among cellular compartments. [ABSTRACT FROM AUTHOR]