Protein Tyrosine Phosphatase CD45 As an Immunity Regulator and a Potential Effector of CAR-T therapy.

The leukocyte common antigen CD45 is a receptor tyrosine phosphatase and one of the most prevalent antigens found on the surface of blood cells. CD45 plays a crucial role in the initial stages of signal transmission from receptors of various immune cell types. Immunodeficiency, autoimmune disorders, and oncological diseases are frequently caused by gene expression disorders and imbalances in CD45 isoforms. Despite extensive research into the structure and functions of CD45, the molecular mechanisms behind its role in transmitting signals from T-cell receptors and chimeric antigen receptors remain not fully understood. It is of utmost importance to comprehend the structural features of CD45 and its function in regulating immune system cell activation to study oncological diseases and the impact of CD45 on lymphocytes and T cells modified by chimeric antigen receptors.

Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

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.

CAR-tropic extracellular vesicles carry tumor-associated antigens and modulate CAR T cell functionality.

Tumor-derived extracellular vesicles (EVs) are active contributors in metastasis and immunosuppression in tumor microenvironment. At least some of the EVs carry tumor surface molecules such as tumor-associated antigens (TAAs) and/or checkpoint inhibitors, and potentially could interact with T cells or CAR T cells. Upon contact with T cells, EVs could alter their phenotype and functions by triggering signaling through TCR or CAR reprogramming them to escape immune response. We hypothesize that EVs that possess TAA on the surface will probably interact with CAR T cells which can recognize and bind corresponding TAA. This interaction between EVs and CAR T cells may change the outcome of CAR T-based cancer immunotherapy since it should affect CAR T cells. Also, EVs could serve as adjuvants and antigenic components of antitumor vaccines. Herein, we isolated EVs from B cell precursor leukemia cell line (pre-B ALL) Nalm-6 and demonstrated that recognition and binding of CD19+EVs with CD19-CAR T cells strongly depends on the presence of CD19 antigen. CD19+EVs induce secretion of pro-inflammatory cytokines (IL-2 and IFN-y) and upregulated transcription of activation-related genes (IFNG, IFNGR1, FASLG, IL2) in CD19-CAR T cells. Tumor necrosis factor receptor superfamily (TNFRSF4 and TNFRSF9) and T-cell exhaustion markers (CTLA4, LAG3, TIM3 and PDCD1LG2) were also upregulated in CD19-CAR T cells after incubation with CD19+EVs. Long-term cultivation of CD19+ or PD-L1+EVs with CD19-CAR T cells led to increased terminal differentiation and functional exhaustion according to elevated expression of PD-1, TIGIT, CD57. In summary, our results suggest that chronic exposure of CD19-CAR T cells to CD19+EVs mediates activation and systemic exhaustion in antigen-specific manner, and this negative effect is accompanied by the impaired cytotoxic activity in vitro.

Engineered Removal of PD-1 From the Surface of CD19 CAR-T Cells Results in Increased Activation and Diminished Survival.

CAR-T cell therapy is the most advanced way to treat therapy resistant hematologic cancers, in particular B cell lymphomas and leukemias, with high efficiency. Donor T cells equipped ex vivo with chimeric receptor recognize target tumor cells and kill them using lytic granules. CAR-T cells that recognize CD19 marker of B cells (CD19 CAR-T) are considered the gold standard of CAR-T therapy and are approved by FDA. But in some cases, CD19 CAR-T cell therapy fails due to immune suppressive microenvironment. It is shown that tumor cells upregulate expression of PD-L1 surface molecule that binds and increases level and signal provided by PD-1 receptor on the surface of therapeutic CAR-T cells. Induction of this negative signaling results in functional impairment of cytotoxic program in CAR-T cells. Multiple attempts were made to block PD-1 signaling by reducing binding or surface level of PD-1 in CAR-T cells by various means. In this study we co-expressed CD19-CAR with PD-1-specific VHH domain of anti-PD-1 nanobody to block PD-1/PD-L1 signaling in CD19 CAR-T cells. Unexpectedly, despite increased activation of CAR-T cells with low level of PD-1, these T cells had reduced survival and diminished cytotoxicity. Functional impairment caused by disrupted PD-1 signaling was accompanied by faster maturation and upregulation of exhaustion marker TIGIT in CAR-T cells. We conclude that PD-1 in addition to its direct negative effect on CAR-induced signaling is required for attenuation of strong stimulation leading to cell death and functional exhaustion. These observations suggest that PD-1 downregulation should not be considered as the way to improve the quality of therapeutic CAR-T cells.

Neutrophil Extracellular Traps (NETs): Opportunities for Targeted Therapy.

Antitumor therapy, including adoptive immunotherapy, inevitably faces powerful counteraction from advanced cancer. If hematological malignancies are currently amenable to therapy with CAR-T lymphocytes (T-cells modified by the chimeric antigen receptor), solid tumors, unfortunately, show a significantly higher degree of resistance to this type of therapy. As recent studies show, the leading role in the escape of solid tumors from the cytotoxic activity of immune cells belongs to the tumor microenvironment (TME). TME consists of several types of cells, including neutrophils, the most numerous cells of the immune system. Recent studies show that the development of the tumor and its ability to metastasize directly affect the extracellular traps of neutrophils (neutrophil extracellular traps, NETs) formed as a result of the response to tumor stimuli. In addition, the nuclear DNA of neutrophils - the main component of NETs - erects a spatial barrier to the interaction of CAR-T with tumor cells. Previous studies have demonstrated the promising potential of deoxyribonuclease I (DNase I) in the destruction of NETs. In this regard, the use of eukaryotic deoxyribonuclease I (DNase I) is promising in the effort to increase the efficiency of CAR-T by reducing the NETs influence in TME. We will examine the role of NETs in TME and the various approaches in the effort to reduce the effect of NETs on a tumor.

[Experimental rationale for the use of a new model of the artificial iridolens stop for reconstructive surgery].

A new-design artificial iridolens stop (ILS) has been constructed, which is monolithic, elastic, universal by possible implantation modalities in relation to the degree of a damage to the anatomy of the anterior segment of the eye, adapted to surgery of minor incisions, and alleviating intra- and postoperative complications. The color spectrum of the haptic part of an ILS yields a good cosmetic effect. The ILS is made from a hydrophobic spatially sutured polymer based on polyoxypropylene. The experimental findings suggest that the proposed ILSs show a chemical and biomedical safety. The stops exert no locally irritant, sensitizing, and toxic effects, they are sterile, apyrogenic and meet the specifications of the items permanently contacting the internal environment of the eye.

[Prognostic value of the content of lipid peroxidation products in tissues at pancreatic necrosis]. / Prognosticheskoe znachenie soderzhaniia produktov lipoperoksidatsii v tkaniakh pri pankreonekroze.

The exact role of lipid peroxidation in pathogenesis of pancreatonecrosis was established by means of studying the level of dienic conjugates and malonic dialdehyde in the intraoperative bioptates of the pancreas, liver, lymph nodes of the lesser omentum in 40 patients as well as in the autopsy material (similar tissues, spleen and bone marrow) in 18 patients dead of destructive pancreatitis. The control group included 10 patients dead of sudden death (normal), group of comparison (those who died of acute myocardium infarction--10 subjects) and of acute surgical abdominal pathology not associated with pancreatitis (10 subjects). Demonstrable activation (6-10 times) of processes of lipid peroxidation in all the bioptates under study took place at pancreatonecrosis as compared to the normal that suggests generalization of lipid peroxidation at this pathology. The level of lipid peroxidation can show the degree of destruction in the pancreas that can be considered as a criterion of the outcome of the disease. In the groups of comparison the level of lipid peroxidation was much lower than at pancreatonecrosis that suggests the pathogenetic role of activation of lipid peroxidation in the development of this disease.

Multirhythmicity generated by slow variable diffusion in a ring of relaxation oscillators and noise-induced abnormal interspike variability.

The deterministic and noise-dependent dynamics of a ring of three Ohmically coupled electronic relaxation oscillators are considered by means of numerical simulations. Each isolated oscillator is described by a set of two ordinary differential equations with very different characteristic times. The emergence of the limit cycle via the Hopf bifurcation results from the N-shaped current-versus-voltage characteristic of the nonlinear resistor. The phase diagram is calculated for a ring of three such oscillators in the presence of small detuning. Special attention is focused on two parameter areas, one near a transition to the homogeneous and the other near the inhomogeneous stable steady state. Along with other nontrivial limit cycles, essentially asymmetrical limit cycles termed dynamic traps may arise in these two areas. A dynamic trap is a regime in which one or two oscillators do not perform full-amplitude oscillations and, correspondingly, do not generate spikes. The interspike interval (ISI) distribution in the presence of noise is calculated as a function of the coupling strength in both areas of the parameter plane. The distributions are extremely polymodal near the homogeneous steady state even if the in-phase limit cycle is dominating. The origins of this abnormal enhancement of ISI variability are discussed in detail. A similar analysis shows that nontrivial periodic attractors are observable in the vicinity of the inhomogeneous stable steady states only if the level of noise is relatively low. In this case, the dominance of the in-phase limit cycle basin results in an almost unimodal distribution of interspike intervals.

[Results of intraocular lens implantation in cataract complicated by medium and high myopia]. / Rezul'taty implantatsii intraokuliarnykh linz pri katarakte, oslozhnennoi miopiei srednei i vysokoi stepeni.

Results of surgical treatment of cataract complicated by medium and high myopia are analyzed. The main group consisted of 90 patients (96 eyes) subjected to extracapsular cataract extraction with implantation of posterior-chamber intraocular lenses and the reference group of 128 patients in whom cataract was extracted without implantation of artificial lens. The study included creation of a mathematical model of myopic eye with estimation of pressure fluctuations in various zones of the eye, developing during transposition of the vitreous during patient's movements (head movements, jumps, falling) and the damping effect of the lens in the ocular system. It was proven by mathematical calculations that transpositions of jelly fractions of the vitreous decreased by 70% in an eye with the lens in comparison with their transposition in aphakia. Clinical studies demonstrated the efficiency of intraocular correction of aphakia in high myopia: implantation of an intraocular lens decreases the risk of detachment of the retina during the postoperative period and helps attain the desired refraction.

[Neurosensory hearing loss in children with acute and chronic otitis media diagnosed by the measurement of short-latency auditory evoked potentials]. / Neirosensornaia tugoukhost' u detei s ostrym i khronicheskim srednim otitom po dannym korotkolatentnykh slukhovykh vyzvannykh potentsialov.

The sensory component of acute otitis media, especially in children, has been poorly studied. In order to identify the importance of ERA for the diagnosis of the neurosensory component of hypoacusis associated with acute otitis media, we examined 56 children. In all the patients ERA were recorded, and in children over 3 years old tonal audiometry was performed. Comparison of ERA and audiographic data showed that in some cases ERA was helpful to reveal the sensory component of impaired hearing which was not seen audiographically. This allowed detection of preclinical stages of perceptive disorders. It is concluded that in young children (younger than 3 years) ERA can help to measure the level of conductive hearing impairment as well as to detect inflammatory changes in the inner ear structures.

[Serological reaction systems using enzyme-labelled immunospecific reagents]. / Sistemy serologicheskikh reaktsii s ispol'zovaniem immunospetsificheskikh reagentov, mechennykh fermentom.

Different serological test systems, based on the use of enzyme-labeled immunospecific reagents and intended for testing the material under study for the presence of Yersinia pestis capsular antigen and antibodies to it, are described. Comparative data on the evaluation of their sensitivity to the antigen and antibodies to it in different schemes of enzyme immunoassays (EIA) are presented. As shown in this investigation, EIA systems for the detection of the antigen and antibodies to it can comprise, at the minimum, the following set of reagents: monoclonal antibodies to the capsular antigen, staphylococcal protein A, and the conjugates of the capsular antigen and monoclonal antibodies with horse-radish peroxidase. The authors have come to the conclusion that the use of the serological test systems can essentially increase the reliability of the assay of any individual sample by EIA techniques.

[Use of monoclonal antibodies in immunoenzyme analysis for demonstrating antigenic monovalency]. / Ispol'zovanie monoklonal'nykh antitel v immunofermentnom analize dlia dokazatel'stva odnovalentnosti antigena.

To prove the monovalence of the antigen a method has been developed consisting of ELISA with the use of monoclonal antibodies in combination with the antibody neutralization test. Yersinia pestis capsular antigen was disintegrated by heating at 100 degrees C for a short time and subsequently passed through a column packed with Sephadex G-50. The portions of the eluate, showing high activity in the antibody neutralization test and low activity in ELISA (the double antibody sandwich scheme), contained mainly the monovalent antigen. This antigen was replaced by the polyvalent antigen from the antibody complex, but if such complex had been previously fixed by treatment with glutaraldehyde, no replacement of the monovalent antigen by the polyvalent one occurred.