Abstract Introduction. Over the past decades, the incidence of malignant blood diseases has been steadily increasing. Unlike many other types of malignant neoplasms, blood cancers often occur at a young age, and are also the main cause of death among all pathologies of the hematopoietic tissue in pediatric patients. The cause of blood cancers is a mutation process in hematopoietic stem cells, which subsequently leads to an increase in the number of tumor clones and the displacement of healthy cells in the niches they occupy, which leads to a number of changes in the blood and bone marrow, including irreversible ones. The aim of this study was to assess the subpopulation composition of pluripotent hematopoietic stem cells in patients with blood cancers and healthy donors. Materials and methods. The study included patients blood cancers (n = 13), including patients with acute lymphoblastic leukemia (ALL) n=3, acute myeloid leukemia (AML) n=3 and patients with multiple myeloma (MM) n=7, and healthy donors (n = 4). The phenotypic composition of hematopoietic CD34+CD38- pluripotent cells was assessed by flow cytometry using the following monoclonal antibodies: CD34 APC (BioLegend, USA), CD38 PE-Cy7 (ElabScience, China), CD45RA PerCP (ElabScience, China), CD90 APC-Cy7 (Cloud-Clone Corp., USA), Lin- (cocktail CD3/14/16/19/20/56) FITC (BioLegend, USA). The following populations were assessed: hematopoietic stem cells (Lin-CD34+CD38-CD45RA-CD90+) and pluripotent progenitors (Lin-CD34+CD38-CD45RA-CD90-). Results. It was shown that in patients with hemoblastoses, the relative number of cells with the Lin-CD34+CD38-CD45RA-CD90+ phenotype increases, which corresponds to hematopoietic stem cells, but not pluripotent progenitor cells. At the same time, the number of pluripotent progenitor cells tends to decrease in acute myeloid leukemia. Conclusions. It was found that in patients with blood cancers, when compared with healthy donors, changes in the subpopulation composition of pluripotent hematopoietic stem cells are observed.

In recent years, hyaluronic acid (HA) has attracted increasing attention as a promising biomaterial for the development of drug delivery systems. Due to its unique properties, such as high biocompatibility, low toxicity, and modifiability, HA is becoming a basis for the creation of targeted drug delivery systems, especially in the field of oncology. Receptors for HA overexpressed in subpopulations of cancer cells and one of them, CD44, is recognized as a molecular marker for cancer stem cells. This review examines the role of HA and its receptors in health and tumors and analyzes existing HA-based delivery systems and their use in various types of cancer. The development of new HA-based drug delivery systems will bring new opportunities and challenges to anti-cancer therapy.

Cucurbiturils are a family of macrocyclic oligomers capable of forming host–guest complexes with various molecules. Due to noncovalent binding to drug molecules and low toxicity, cucurbiturils has been extensively investigated as potential carriers for drug delivery. However, the immune system’s interactions with different drug carriers, including cucurbiturils, are still under investigation. In this study, we focused on cucurbiturils’ immunosafety and immunomodulation properties in vivo. We measured blood counts and lymphocyte subpopulations in blood, spleen, and bone marrow, and assessed the in vivo toxicity to spleen and bone marrow cells after intraperitoneal administration to BALB/c mice. When assessing the effect of cucurbit[6]uril on blood parameters after three intraperitoneal injections within a week in laboratory animals, a decrease in white blood cells was found in mice after injections of cucurbit[6]util, but the observed decrease in the number of white blood cells was within the normal range. At the same time, cucurbit[7]uril and cucurbit[8]uril did not affect the leukocyte counts of mice after three injections. Changes in the number of platelets, erythrocytes, and monocytes, as well as in several other indicators, such as hematocrit or erythrocyte volumetric dispersion, were not detected. We show that cucurbiturils do not have immunotoxicity in vivo, with the exception of a cytotoxic effect on spleen cells after сucurbit[7]uril administration at a high dosage. We also evaluated the effect of cucurbiturils on cellular and humoral immune responses. We founded that cucurbiturils in high concentrations affect the immune system in vivo, and the action of various cucurbiturils differs in different homologues, which is apparently associated with different interactions in the internal environment of the body.

Immunological safety of nanoparticles is an urgent problem for development of drug delivery systems used as the basis for creating these systems. Cucurbit[n]urils (CB[n]) are molecular nanocontainers that can encapsulate various drugs and serve as the basis for delivery systems. Cucurbiturils are low-toxic compounds: under in vitro conditions, they exhibit weak immunomodulatory properties, without sufficient immunotoxicity. The aim of the present study was to evaluate the in vivo effects of cucurbiturils on the number and differential leukocyte counts in peripheral blood. BALB/c mice aged 2-4 months were used in the work. For this study, cucurbiturils were diluted in phosphate-buffered saline and administered to laboratory animals intraperitoneally (three times a week). When evaluating the effect of cucurbit[7]uril on blood parameters of the animals after three intraperitoneal injections weekly, no statistically significant changes were registered. However, at first administration, the animals showed a slight increase in relative number of lymphocytes after the first injection of CB[6], and an increased proportion of neutrophils after the first administration of CB[8]. Meanwhile, the proportions of lymphocytes and neutrophils were within normal ranges after the 2nd and 3rd injections of cucurbiturils, and did not show any significant differences against the controls. Moreover, the impact upon the subpopulation composition of peripheral blood lymphocytes in laboratory animals was assessed. After injection of CB[6], there was a decrease in T lymphocytes in the peripheral blood, along with increased proportion of CD19+B lymphocytes compared with the controls. CB[7] and CB[8] injections did not affect the subpopulation profile of peripheral blood lymphocytes. We have found that intraperitoneal administration of CB[n] did not affect the blood parameters of laboratory animals, thus, probably suggesting the in vivo safety of these compounds. At the same time, CB[6] is able to exert a stimulating effect on humoral immunity by increasing relative contents of B lymphocytes.

Short regulatory oligonucleotides are considered prospective tools for immunotherapy. However, they require an adequate carrier to deliver potential therapeutics into immune cells. Herein, we explore the potential of polycationic dendrimers as carriers for microRNAs in peripheral blood mononuclear cells of healthy donors. As an oligonucleotide cargo, we use a synthetic mimic and an inhibitor of miR-155, an important factor in the development and functioning of immunocompetent cells. Dendrimers bind microRNAs into low-cytotoxic polyelectrolyte complexes that are efficiently uptaken by immunocompetent cells. We have shown these complexes to affect the number of T-regulatory cells, CD14+ and CD19+ cell subpopulations in non-activated mononuclear cells. The treatment affected the expression of HLA-DR on T-cells and PD-1 expression on T- and B-lymphocytes. It also affected the production of IL-4 and IL-10, but not the perforin and granzyme B production. Our findings suggest the potential of dendrimer-mediated microRNA-155 treatment for immunotherapy, though the activity of microRNA-dendrimer constructions on distinct immune cell subsets can be further improved.

Glioblastoma is a rapidly progressing tumor quite resistant to conventional treatment. These features are currently assigned to a self-sustaining population of glioblastoma stem cells. Anti-tumor stem cell therapy calls for a new means of treatment. In particular, microRNA-based treatment is a solution, which in turn requires specific carriers for intracellular delivery of functional oligonucleotides. Herein, we report a preclinical in vitro validation of antitumor activity of nanoformulations containing antitumor microRNA miR-34a and microRNA-21 synthetic inhibitor and polycationic phosphorus and carbosilane dendrimers. The testing was carried out in a panel of glioblastoma and glioma cell lines, glioblastoma stem-like cells and induced pluripotent stem cells. We have shown dendrimer-microRNA nanoformulations to induce cell death in a controllable manner, with cytotoxic effects being more pronounced in tumor cells than in non-tumor stem cells. Furthermore, nanoformulations affected the expression of proteins responsible for interactions between the tumor and its immune microenvironment: surface markers (PD-L1, TIM3, CD47) and IL-10. Our findings evidence the potential of dendrimer-based therapeutic constructions for the anti-tumor stem cell therapy worth further investigation.

Tumor cells with stem cell properties are considered to play major roles in promoting the development and malignant behavior of aggressive cancers. Therapeutic strategies that efficiently eradicate such tumor stem cells are of highest clinical need. Herein, we performed the validation of the polycationic phosphorus dendrimer-based approach for small interfering RNAs delivery in in vitro stem-like cells as models. As a therapeutic target, we chose Lyn, a member of the Src family kinases as an example of a prominent enzyme class widely discussed as a potent anti-cancer intervention point. Our selection is guided by our discovery that Lyn mRNA expression level in glioma, a class of brain tumors, possesses significant negative clinical predictive value, promoting its potential as a therapeutic target for future molecular-targeted treatments. We then showed that anti-Lyn siRNA, delivered into Lyn-expressing glioma cell model reduces the cell viability, a fact that was not observed in a cell model that lacks Lyn-expression. Furthermore, we have found that the dendrimer itself influences various parameters of the cells such as the expression of surface markers PD-L1, TIM-3 and CD47, targets for immune recognition and other biological processes suggested to be regulating glioblastoma cell invasion. Our findings prove the potential of dendrimer-based platforms for therapeutic applications, which might help to eradicate the population of cancer cells with augmented chemotherapy resistance. Moreover, the results further promote our functional stem cell technology as suitable component in early stage drug development.

It is well known that the most important feature of adaptive immunity is the specificity that provides highly precise recognition of the self, altered-self, and non-self. Due to the high specificity of antigen recognition, the adaptive immune system participates in the maintenance of genetic homeostasis, supports multicellularity, and protects an organism from different pathogens at a qualitatively different level than innate immunity. This seemingly simple property is based on millions of years of evolution that led to the formation of diversification mechanisms of antigen-recognizing receptors and later to the emergence of a system of presentation of the self and non-self antigens. The latter could have a crucial significance because the presentation of nearly complete diversity of auto-antigens in the thymus allows for the "calibration" of the forming repertoires of T-cells for the recognition of self, altered-self, and non-self antigens that are presented on the periphery. The central role in this process belongs to promiscuous gene expression by the thymic epithelial cells that express nearly the whole spectrum of proteins encoded in the genome, meanwhile maintaining their cellular identity. This complex mechanism requires strict control that is executed by several transcription factors. One of the most important of them is AIRE. This noncanonical transcription factor not only regulates the processes of differentiation and expression of peripheral tissue-specific antigens in the thymic medullar epithelial cells but also controls intercellular interactions in the thymus. Besides, it participates in an increase in the diversity and transfer of presented antigens and thus influences the formation of repertoires of maturing thymocytes. Due to these complex effects, AIRE is also called a transcriptional regulator. In this review, we briefly described the history of AIRE discovery, its structure, functions, and role in the formation of antigen-recognizing receptor repertoires, along with other transcription factors. We focused on the phylogenetic prerequisites for the development of modern adaptive immunity and emphasized the importance of the antigen presentation system.

In this review, we described the structure and organization of antigen-recognizing repertoires of B and T cells from the standpoint of modern immunology. We summarized the latest advances in bioinformatics analysis of sequencing data from T and B cell repertoires and also presented contemporary ideas about the mechanisms of clonal diversity formation at different stages of organism development. At the same time, we focused on the importance of the allelic variants of the HLA genes and spectra of presented antigens for the formation of T-cell receptors (TCR) landscapes. The main idea of this review is that immune equilibrium and proper functioning of immunity are highly dependent on the interaction between the recognition and the presentation landscapes of antigens. Certain changes in these landscapes can occur during life, which can affect the protective function of adaptive immunity. We described some mechanisms associated with these changes, for example, the conversion of effector cells into regulatory cells and vice versa due to the trans-differentiation or bystander effect, changes in the clonal organization of the general TCR repertoire due to homeostatic proliferation or aging, and the background for the altered presentation of some antigens due to SNP mutations of MHC, or the alteration of the presenting antigens due to post-translational modifications. The authors suggest that such alterations can lead to an increase in the risk of the development of oncological and autoimmune diseases and influence the sensitivity of the organism to different infectious agents.

Rheumatoid arthritis (RA) is a chronic disease of connective tissue caused by intolerance to self-antigens. Regulatory T cells (Tregs) are key players in maintaining autotolerance through a variety of suppressor mechanisms. RA is generally believed to develop due to disorders in Tregs; however, there is no consensus on this issue. Thus, the present study focused on phenotypical analysis of Treg cells and their ability to suppress CD4+ and CD8+ cell proliferation. The present study used peripheral blood samples from 21 patients with RA and 22 healthy donors. The CD25+FoxP3+ subpopulation of Tregs was analyzed using flow cytometry to evaluate the expression of CTLA-4, PD-L1, HLA-DR, CCR4, CD86 and RORyt. Tregs suppressor activity was calculated in terms of suppression of the proliferation of CD4+ and CD8+ lymphocytes in vitro. Suppressor activity of the total Treg population did not differ between patients with RA and healthy donors. However, the patients had elevated CD25loFoxP3+ levels and lower CD25hiFoxP3+ levels; in addition, they had more activated Tregs expressing PD-L1, HLA-DR, CCR4 and CD86. The surface expression of CTLA-4 was below the reference level. The patients also had transitional FoxP3+RORyt+ cells and elevated CD4+RORyt+ levels, which were highly correlated with disease activity. These results show that in RA, Treg cells are activated and have an immunosuppressive activity. However, it is the transitional FoxP3+RORyt+ cells and increased CD4+RORyt+ percentages in peripheral blood that appear to be associated with the pathological conversion of some Treg cells into Th-17. This process appears to be key in RA pathogenesis.

The crystal structure of cucurbituril (CB[n]) resembles that of a pumpkin or a barrel with the top and bottom portals formed by the oxygen atoms of the highly polarized carbonyl groups. This configuration is unique and allows CB[n] to accommodate many different molecules in its cavity through ?guest-host? binding involving noncovalent bonds. Supramolecular CB[n] containers, in particular CB[7], are promising transport systems for various therapeutic agents, which in turn, open up new opportunities for applications of long-used drugs. Some of these drugs, the pharmacological properties of which can be improved, are platinum(II) compounds, known for their cytostatic activity. The impact of CB[n] encapsulation on the biological properties of drugs has not been widely studied, but it is of great interest at present. In this article, we review current research on the effect of CB [7] complexation on the properties of platinum(II) compounds to evaluate the progress achieved in this area.

The most effective method of treating allergic diseases, aimed not at relieving symptoms, but at eliminating the cause of the disease, is allergen-specific immunotherapy (AIT). To reduce the risk of side effects and improve the delivery of allergens to the mucosa, various delivery systems, such as liposomes, dendrimers, nanoparticles, etc., can be used. To date, there are data on the creation of delivery systems based on glycyrrhizic acid (GA) and its derivatives, but such a delivery system has not been used for allergen-specific therapy until now. It is also known that GA has an anti-inflammatory effect, shifts the balance towards Th1, and increases the number of Treg cells, meaning that it could potentially enhance the anti-allergic effect of AIT and reduce the risk of unwanted side effects. Thus, the study of the immunomodulatory effect of the supramolecular complexes (micelles) of GA with extracts of allergens holds promise for the development of new drugs for AIT.

Currently, cucurbiturils are being actively researched all over the world. Research is focused on the ways of improving the solubility and selectivity of cucurbiturils, increasing the stability of the complexes with other particles in various media and enhancing their ability to bind and release various substances. The most significant area of our research is the assessment of safety, studying the biological properties and synergistic effects of cucurbiturils during complexation with drugs. In this article, the hemocompatibility of erythrocytes and leukocytes with cucurbiturils was investigated. We demonstrated that cucurbiturils have no cytotoxic effect, even at high concentrations (1 mM) and do not affect the viability of PBMCs. However, cucurbiturils can increase the level of the early apoptosis of lymphocytes and cucurbit[7]uril enhances hemolysis in biologically relevant media. Despite this, cucurbiturils are fairly safe organic molecules in concentrations up to 0.3 mM. Thus, we believe that it will become possible to use polymer nanostructures as drug delivery systems in clinical practice, since cucurbiturils can be modified to improve pharmacological properties.

The most important functions of the immune system are the guarantee of multicellularity, the maintenance of genetic homeostasis, and the protection of the organism from various infectious agents. The maintenance of homeostasis within the immune system also plays an important role in the normal functioning of the organism and is realized through several mechanisms. For example, after the resolution of the immune response, the excessive amount of lymphocytes is eliminated via apoptosis due to the competition for the survival factors and because of the lack of specific stimulation, and the excessive amount of antibodies is corrected by idiotype–anti-idiotype interactions. The restoration and maintenance of the lymphocyte count are performed due to the migration from the bone marrow and thymus and the process of homeostatic proliferation that plays the most important role (after the involution of the thymus) for T-lymphocytes. Because of the non-uniformity of the definitions that are met in the published literature, the authors provide the definition of the process of homeostatic proliferation of T-lymphocytes that will be used further in the text. Homeostatic proliferation (HP) is a physiological process to restoration of the peripheral T-lymphocyte pool after lymphopenia of any etiology through the antigen-specific proliferation of lymphocytes under the influence of IL-7 and IL-15, which can acquire pathological features depending on the severity of lymphopenia. Homeostatic proliferation can be conventionally divided into a fast and slow type depending on its intensity. Although such division is highly conventional, there are certain physiological peculiarities of fast and slow HP that can influence the functioning of the immune system, change the T-cell receptor landscape, and lead to the development of pathologies. It is worth noting that fast HP predominantly involves memory effector T-cells, while slow HP also influences naive T-cells. In the present review, the authors discuss the most important physiological and pathological aspects of the homeostatic proliferation of T-lymphocytes.

Homeostatic proliferation (HP) is a physiological process that reconstitutes the T cell pool after lymphopenia involving Interleukin-7 and 15 (IL-7 and IL-15), which are the key cytokines regulating the process. However, there is no evidence that these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the functional activity of Tregs stimulated by HP cytokines from patients with rheumatoid arthritis as compared with that of those from healthy donors. Since T cell receptor (TCR) signal strength determines the intensity of HP, we imitated slow HP using IL-7 or IL-15 and fast HP using a combination of IL-7 or IL-15 with anti-CD3 antibodies, cultivating Treg cells with peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio. We used peripheral blood from 14 patients with rheumatoid arthritis and 18 healthy volunteers. We also used anti-CD3 and anti-CD3 + IL-2 stimulation as controls. The suppressive activity of Treg cells was evaluated in each case by the inhibition of the proliferation of CD4+ and CD8+ cells. The phenotype and proliferation of purified CD3+CD4+CD25+CD127lo cells were assessed by flow cytometry. The suppressive activity of the total pool of Tregs did not differ between the rheumatoid arthritis and healthy donors; however, it significantly decreased in conditions close to fast HP when the influence of HP cytokines was accompanied by anti-CD3 stimulation. The Treg proliferation caused by HP cytokines was lower in the rheumatoid arthritis (RA) patients than in the healthy individuals. The revealed decrease in Treg suppressive activity could impact the TCR landscape during lymphopenia and lead to the proliferation of potentially self-reactive T cell clones that are able to receive relatively strong TCR signals. This may be another explanation as to why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease in Treg proliferation under IL-7 and IL-15 exposure can lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis in the case of lymphopenia.

Cucurbit[7]uril (CB[7]) is a molecular container that may form host–guest complexes with platinum(II) anticancer drugs and modulate their efficacy and safety. In this paper, we report our studies of the effect of CB[7]–oxaliplatin complex and the mixture of CB[7] and carboplatin (1:1) on viability and proliferation of a primary cell culture (peripheral blood mononuclear cells), two tumor cell lines (B16 and K562) and their activity in the animal model of melanoma. At the same time, we studied the impact of platinum (II) drugs with CB[7] on T cells and B cells in vitro. Although the stable CB[7]–carboplatin complex was not formed, the presence of cucurbit[7]uril affected the biological properties of carboplatin. In vivo, CB[7] increased the antitumor effect of carboplatin, but, at the same time, increased its acute toxicity. Compared to free oxaliplatin, its complex with CB[7] shows a greater cytotoxic effect on tumor cell lines B16 and K562, while in vivo, the effects of the free drug and encapsulated drug were comparable. However, in vivo studies also demonstrated that the encapsulation of oxaliplatin in CB[7] lowered the toxicity of the drug.

Homeostatic proliferation (HP) is a physiological process to reconstitute the T-cell pool after lymphopenia with IL-7 and IL-15 being the key cytokines regulating the process. However, there is no evidence whether these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the proliferation rate and function of Tregs stimulated by IL-7 and IL-15 in patients with rheumatoid arthritis (RA) compared to healthy donors (HD). The study used peripheral blood from 14 RA patients and 18 HD. Proliferation of purified CD3CD4CD25CD127 cells was assessed by flow cytometry using CFSE. Tregs were stimulated by anti-CD3, IL-7, IL-15, IL-7, or IL-15 combined with anti-CD3, and by IL-2+anti-CD3, and their functional activity was evaluated in each case by CD4 and CD8 cells proliferation inhibition. The suppressive activity of peripheral Tregs did not differ between RA and HD; however, it significantly decreased when IL-7 or IL-15 were applied together with strong TCR stimulation with anti-CD3 antibodies. Herewith Treg proliferation caused by IL-7 and IL-15 was lower in RA than in HD. The revealed decrease in Treg suppressive activity can lead to the proliferation of potentially self-reactive T-cell clones, which can receive relatively strong TCR signals. This may be another explanation of why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease of Treg proliferation under IL-7 and IL-15 may lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis.

Cucurbiturils (CB[n]s) are nanoscale macrocyclic compounds capable of encapsulating a molecule or part of a molecule by forming host–guest complexes. Integration of drugs with CB[n] is used for the following purposes: controlling clearance; protection of the drug from biodegradation; targeted delivery to specific organs, tissues, or cells; reduction of toxicity; and improving solubility. One of the major problems encountered in the application of new drug delivery systems is lack of knowledge of their biological properties. CB[n], unlike many other often toxic nanoparticles, has extremely low toxicity, even at high doses. However, many aspects of the biological actions of these nanoscale cavitands remain unclear, including the immunotropic properties. In this study, we investigated the immunotoxicity and immunomodulation properties of CB[n]. It was found that CB[7] and CB[6] did not decrease the viability of mononuclear cells at all tested concentrations from 0.1–1 mM. Overall, the results indicated an immunomodulatory effect of different concentrations of CB[n]. In the case of a longer cultivation time, CB[n] had an immunostimulating effect, which was indicated by an enhancement of the proliferative activity of cells and increased expression of HLA-DR on lymphocytes.