Dynamics of morphological, immunological and histological changes in microsporіа in guinea pigs

Microsporіа affect different species of animals and humans. The high contagiousness of the pathogen determines the relevance of research into this disease. Microsporum canis is the pathogen that most often causes microsporia. Weakened functions of the immune system and violation of the epithelial barrier of the skin are a favourable factor that causes microspores. The main source of infection is cats, which are involved in the storage and transmission of the pathogen. To clarify the dynamics of morphological, immunological and histological changes in microsporia, blood and skin studies of guinea pigs infected with M. canis were carried out. The animals were divided into two groups of 6 guinea pigs (healthy and sick). Test material (blood and skin) was taken from clinically healthy and sick animals 21 and 42 days after infection. The number of erythrocytes and leukocytes was determined by counting them in the Goryaev chamber, the hemoglobin content – by the method of cyanide hemoglobin. The leukogram was derived based on the counting and differentiation of 200 leukocyte cells in blood smears. Material for histological examination (pieces of skin) was fixed in 10–12% cooled solution of neutral formalin, followed by pouring in paraffin according to the scheme proposed by G. A. Merkulov. The obtained results demonstrated that leukocytosis developed in guinea pigs with microsporia on the 21st and 42nd days; the number of rod-shaped neutrophils increased, that of segmental neutrophils decreased, and that of ESR increased. The immune response to the course of microsporia was manifested in an increase in the percentage of T-lymphocytes, T-suppressors and a decrease in T-helper cells and an increase in T-killers compared with healthy animals. Histological examination showed that on the 21st day after infection, hyphae and spores of the fungus M. canis were localized in the skin. There is swelling of the dermis, stratification of collagen fibers and the accumulation of inflammatory infiltrates around the hair follicles. On the 42nd day, the infiltration spread and dystrophic changes in the skin occurred in the form of desquamation of the epidermis and the formation of acanthosis and hyperkeratosis on the surface of the dermis. The conducted research will allow further assessment of the course of microsporia under the action of various drugs and help establish the most effective method of treatment.

Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells

Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

Artificial Microvesicles Isolated from Mesenchymal Stem Cells with IL2 Overexpression Activate CD8+ T-Killers to Kill Triple Negative Breast Cancer Cells

Almost all human cells release extracellular vesicles (EVs) participating in intercellular communication. EVs are rounded structures surrounded by the cytoplasmic membrane, which embody cytoplasmic contents of the parent cells. Since the orientation of surface receptors persists during EV formation, it is EVs that are of interest as a therapeutic tool for cell-free therapy that has targeted delivery. The ideal cell type for the EV production are mesenchymal stem cells (MSCs) as they exhibit a homing behaviour to tumor niches. In this study, human MSCs were isolated from adipose tissue (hADSCs) and genetically modified to overexpress interleukin-2 (IL2) (hADSCs-IL2) or red fluorescent protein (hADSCs-RFP). Extracellular vesicle release from native and genetically modified hADSCs was induced by cytochalasin B treatment. Size and immunophenotype of isolated Cytochalasin B induced microvesicles (CIMVs) were analysed using flow cytometry. To evaluate immunomodulating properties of microvesicles from native (native CIMVs) or genetically modified (CIMVs-IL2) hADSCs, peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and co-cultured with CIMVs (50 μg/ml) for 72 hours. Activation of PBMC populations was determined by flow cytometry using staining with antibodies specific for surface markers of various human immune cell populations. To analyze the antitumor activity of PBMCs after interaction with CIMVs-IL2, activated PBMCs were transferred to triple negative breast cancer cells MDA-MB-231 which were pre-seeded on 16-well xCelligence plate (3 × 103 cells per well) or 6-well plate (5 × 104 cells per well) in 1:3 ratio (tumor cells:PBMCs). Proliferative activity of tumor cells was evaluated using an RTCA xCelligence instrument (ACEA Biosciences, USA) for 72 hours. Tumor cell viability was assessed 24 hours after culturing with PBMCs using the APC Annexin V Apoptosis Detection Kit with PI (#640932, Biolegend, USA) on FACS Aria III flow cytometer (BD, USA). Isolated CIMVs were mostly 50-200 nm in diameter and positive for CD44, CD90 and CD105 MSC surface markers, but CD29 and CD73 levels were significantly decreased. The number of activated T-killers (CD3+CD8+CD4-CD38+HLA-DR+) was significantly increased after incubation with CIMVs-IL2 (205.6 ± 4.5%) compare to native PBMCs (100.0 ± 9%) and PBMCs incubated with native CIMVs (116.7 ± 4.5%) (Fig. 1A). The number Th1 cells was decreased in the sample of PBMCs incubated with CIMVs-IL2 (92.3 ± 1.6%) compared with native PBMCs (100.0 ± 1%) (Fig. 1B), while the number of Th2 cells (109.0 ± 0.1%) was increased compared to native PBMCs (100.0 ± 1%) (Fig. 1C), number of Tregs was unchanged (Fig. 1D). After the addition of PBMCs, activated with CIMVs-IL2, the proliferation of MDA-MB-231 cells has been significantly decreases. Twenty four hours after the addition of CIMVs-IL2-activated PBMCs the proliferation of MDA-MB-231 was decreased 4-fold (to 25.0 ± 5.6%) compared to native MDA-MB-231 (100.0 ± 2.9%) and MDA-MB-231 incubated with native PBMCs (110.8 ± 8.7%) and PBMCs after native CIMVs (103.7 ± 2.4%) (Fig. 1E,G). This decrease in proliferation is likely due to the fact that part of the tumor cells underwent apoptosis and then died which is confirmed by the MDA-MB-231 cell viability after 24 hours of cultivation with PBMCs activated with CIMVs-IL2 (Fig. 1F). The number of healthy MDA-MB-231 cells after 24 hours of cultivation with PBMCs after CIMVs-IL2 was reduced by 35% (46.1 ± 3.7%) compared to native MDA-MB-231 (78.2 ± 2.1%) and MDA-MB-231 incubated with native PBMCs (70.1 ± 2.8%) and PBMCs after native CIMVs (62.1 ± 3.2%). Viable MDA-MB-231 cells in the sample of PBMCs after CIMVs-IL2 continue to proliferate, however their proliferative activity remains reduced (41.2 ± 4.7%) after 72 hours of cultivation compared to native MDA-MB-231 (100.0 ± 21.0%) and MDA-MB-231 incubated with native PBMCs (109.7 ± 2.5%) and PBMCs after native CIMVs (82.5 ± 20.5%) (Fig. 1H). The use of CIMVs-IL2 can be effective in the treatment of triple negative breast cancer, since CIMVs-IL2 are able to activate and stimulate the proliferation of T-killers, which in turn are able to induce apoptosis in breast cancer cells. However, further studies of CIMVs efficiency in animal tumor models are required. This study was supported by the Russian Science Foundation grant 18-74-10044 and the Russian Government Program of Competitive Growth of KFU. Figure Disclosures No relevant conflicts of interest to declare.

The Role of Recombinant Human Cyclophilin A in the Antitumor Immune Response

Cyclophilin A (CypA) is a multifunctional protein that exhibits an isomerase activity and exists in the intracellular and secretory forms. Secretory CypA promotes regeneration of the hematopoietic and the immune systems of an organism by stimulating stem cell migration from the bone marrow. New approaches based on CypA are currently being developed for the treatment of limb ischemia, neutralization of the side effects of Cyclosporine A (CsA) therapy, etc. However, the role of CypA in the antitumor immune response is still unexplored. In this work, we used the model experimental system of lymphoma EL-4 rejection in B10.D2(R101) mice and showed that recombinant human CypA (rhCypA) stimulates the antitumor immune response via early recruitment of granulocytes to the tumor cell localization site and rapid accumulation of effector T-killers

Evaluation of clinical efficacy of Derinat® in form of spray in practice of district therapist

The widespread worldwide spread of acute respiratory diseases is an urgent problem in health care. Expressed polyetiology of respiratory diseases does not allow to limit the use of specific vaccine preparations and dictates the need to use to combat them a variety of non-specific means that stimulate the natural resistance of the human body. The main pharmacological action of sodium deoxyribonucleate is the stimulation of phagocytic activity of T-helpers and T-killers, increasing the functional activity of neutrophils and monocytes/ macrophages, providing regeneration and repair processes in the epithelial component of antiviral protection of the body. Based on the above, the study of the clinical efficacy of Derinat® in the form of spray in the treatment of acute respiratory viral infections is relevant.