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.

Cellular homeostasis in the body is known to be maintained by the processes of cell proliferation and death, whereas apoptosis is the most frequent and physiological, “silent” mechanism of cell elimination. It has been currently shown that the process of apoptosis traditionally considered an autonomous event, has a pronounced non-autonomous effect on migration, proliferation, and death of the neighboring cells. This work was based on the data on impaired programmed death of mononuclear cells from the patients with rheumatoid arthritis (RA) leading to the evolving autoimmune inflammation. The aim of this study was to evaluate effector molecules exerting autonomous and non-autonomous influence of T cell apoptosis under the conditions of “cell neighborhood” in cell cultures of healthy people and RA patients. The studies were performed with blood samples of RA patients and healthy women of comparable age. These experiments were performed in order to assess the levels of main molecules mediating the in vitro receptor and mitochondrial apoptosis of T lymphocytes. In previous studies, using the original “cell neighborhood” model, no differences were found in parameters of early and late activation apoptosis between the groups of donors and RA patients. At the same time, 1-week incubation in apoptotic cultures of the patients was followed by significantly increased number of viable cells carrying the proliferation marker Ki-67. Different results of in vitro apoptosis induction in cultures under similar conditions of “cell neighborhood” in healthy people and patients with RA have revealed the importance of main effector molecules of apoptosis in the studied groups. In this study, we have revealed low potential of the receptor pathway for apoptosis activation in healthy people, due to suppression of TNFα production during cell incubation under the conditions of “cell neighborhood”, and in RA patients due to initially low TNFα in supernatants which did not change over time and in various incubation variants, along with low content of initiating caspase 8 in both groups. Significant suppression of effector molecules of mitochondrial pathway of apoptosis activation, i.e., Bcl-2 anti-apoptotic factor and p53 transcription factor was detected in cultures of apoptotic cells, as well as mixtures of proliferating and apoptotic cells under the conditions of “cell neighborhood” in RA patients. The amounts of these molecules did not change in healthy persons. At the same time, no differences in these molecules were found between individual variants of cell cultures from the patients with RA and healthy people. The both studied groups were characterized by a significant activation of IL-4 and IL-6 production, i.e., the cytokines with autonomous and non-autonomous protective and reparative properties, Hence, one may conclude that high levels of these cytokines had different effects in cell cultures under the conditions of “cell neighborhood”. Incubation of cells from healthy people under suboptimal conditions was associated with maintaining the balance of proliferation and apoptosis, whereas, in cell cultures of RA patients, this balance caused activation of proliferation processes, being accompanied by an increase in the number of living cells in apoptotic cultures.

Известно, что дефекты программированной гибели лимфоцитов периферической крови могут быть причиной аутотолерантности и хронической персистенции воспаления при ревматоидном артрите. По последним данным, процесс апоптоза не является автономным – избыточное скопление клеток в совокупности с обедненным составом среды приводит к скученности клеточного сообщества и влиянию уже ушедших в апоптоз клеток на окружающие клетки посредством морфогенетического повреждения в процессе воздействия механических сил. Такие эффекты называют компенсаторной пролиферацией, или апоптоз-индуцированной пролиферацией. Внеклеточные везикулы (эктосомы и апоптотические тельца) также оказывают негативное воздействие на культивируемые клетки, инициируя запуск программы апоптотической гибели. В свою очередь, апоптоз также может не автономно контролироваться соседними клетками.Нами были проведены исследования, позволившие оптимизировать методы, направленные на инициацию апоптотической гибели клеток и изучение воздействия апоптотического окружения на аутологичные клетки, находящиеся в физиологических условиях. Подбор условий в совокупности с методом флуоресцентного маркирования лимфоцитов и последующим раздельным анализом их на проточном цитофлуориметре позволили оценить параметры раннего апоптоза субпопуляций Т-лимфоцитов периферической крови при ревматоидном артрите. У пациентов с ревматоидным артритом in vitro установлена выраженная готовность к раннему апоптозу первично (CFSE) и вторично (CFSE+) индуцированных Т-лимфоцитов, стимулированных aCD3-антителами, как относительно исходного уровня апоптоза, так и...

The aim of the study was to investigate the features of T-lymphocyte apoptosis induced by components of autologous apoptotic cultures in vitro in norm and rheumatoid arthritis in the context of «cellular neighborhood».Materials and methods. Subjects of the study were blood samples of patients with rheumatoid arthritis (RA) and healthy women of comparable age. Developed protocol allowed to differentially evaluate the parameters of proliferation, early and late stages of apoptosis in the «primary» (CFSE-) and «secondary» (CFSE+) induced apoptotic T-lymphocyte cultures. It was estimated the effect of cellular and humoral components of unstimulated, anti-CD3- and dexamethasone-stimulated cells under the conditions of overcrowding and depleted culture media on autologous lymphocytes, cultured under physiological conditions, in norm and RA.Results. Comparative qualitative analysis revealed the features of the processes of T-lymphocyte apoptosis in norm and pathology. Also, the parameters of early and late stages of apoptosis of a «primary» induced culture and «secondary» induced cells after transferring the cellular and humoral components of apoptotic cultures did not differ significantly either initially or during culturing in both investigated groups. But it was a significant increase in the amount of living T-cells in «primary»-induced unstimulated and dexamethasone-stimulated RA patients’ cultures compared to similar donors’ cultures.Conclusion. There was no difference between stimulated with anti-CD3 antibodies cells and the «secondary» induced cultures. Taking into account the absence of significant differences in the parameters of activation apoptosis, the increased number of living cells in RA patients’ cultures relative to donors’ is evidence of contribution of non-autonomous apoptosis effects to cellular homeostasis in RA.

The process of apoptosis is known that play an important role in cellular homeostasis, and the altered cell death may lead to development of pathological disorders. Evolving autoimmune conditions, in particular, rheumatoid arthritis, are associated with decreased rates of apoptosis as a form of programmed cell death. The aim of this study was to evaluate expression of activation and proliferation markers on T lymphocytes during initiation of apoptotic cell death under the conditions of “cell neighborhood” in healthy individuals and patients with rheumatoid arthritis. Patients and methods. The study was performed with blood samples of the patients with rheumatoid arthritis (RA) and healthy women of comparable age. During the study, we conducted experiments aimed to identify the in vitro influence of non-stimulated apoptosis-induced cells, as well as aCD3^-and dexamethasone (Dexa)-stimulated apoptosis-induced cells upon autologous T lymphocytes cultured under physiological conditions. Development of a “cell neighborhood” model, i.e., co-cultures of CFSE^-T cells subjected to incubation under crowding condition and depletion of the culture medium which is the most physiological variant of apoptosis activation, and CFSE⁺autologous cells placed in the complete culture medium, has revealed some relationships. We have revealed an opportunity of secondary induction of early and late apoptosis by means of humoral and cellular components of autologous cell culture subjected to activation apoptosis. We determined the features of apoptosis in unstimulated, as well as aCD3^-and dexamethasone-stimulated cultures, compared with controls. There were no differences in these parameters of apoptosis between RA patients and healthy people for all variants of cultures. An increased proportion of viale cells was found in the CFSE^-culture of patients with RA when compared to donors. The donor group had more lymphocytes with activation parameters CD25⁺, CD69⁺and low level of proliferation marker Ki-67 than patients. In contrast to healthy, the RA patients demonstrated a significantly increased expression of Ki 67 in T lymphocytes when co-culturing CFSE^-and CFSE⁺cells. An increased number of living cells in apoptotic cultures of patients with RA relative to healthy people, in absence of significant differences in the parameters of apoptosis and activation markers in dynamics, as well as pattern of changes in the Ki-67⁺cell contents suggested a contribution of the non-autonomous effects of apoptosis to cellular homeostasis in RA patients.