Angelina Vladimirovna Pakhomova

Liver cirrhosis, a widespread disease worldwide, can be the result of various causes, such as hepatitis, obesity, and excessive alcohol consumption. The effectiveness of liver cirrhosis therapy is low, as the prescribed medications do not promote the regression of fibrosis or the regeneration of hepatocytes. Therefore, the research and development of new treatment methods for liver cirrhosis remains a pressing task. It is also important to consider age-related features in the course of the disease and responses to therapy. Purpose of the study was to investigate the therapeutic effectiveness of multicomponent therapy for liver cirrhosis in male Wistar rats of different ages. Material and methods . Experiments were performed on 12- and 36-week-old male Wistar rats. Liver cirrhosis was induced by 40% oil solution of CCl ₄ to animals that received 5% ethanol solution and glucose which were freely available throughout the experiment. Polytherapy was carried out according to the following scheme: ketanserin from 85 to 95 days after the first administration of CCl ₄ , reserpine on days 96 and 100, endothelial growth factor from 96 to 102 days, hepatocyte growth factor on day 100. The content of total and direct bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and cholesterol was measured in the blood serum. On histological preparations of the liver, inflammation, fibrosis and the degree of liver damage were assessed, the number of capillaries and the area of connective tissue were calculated. Results . With a course of administration of CCl ₄ and alcohol in rats of both age groups, the development of multilobular cirrhosis, a decrease in the number of sinusoidal capillaries, an increase in the area of connective tissue were noted in the liver tissue and the levels of serum alkaline phosphatase, AST and ALT. Polytherapy of animals with the liver cirrhosis led to a decrease in the intensity of inflammatory infiltration of the liver parenchyma, a decrease in the area of connective tissue in the liver (by 22 % in 12-week-old rats, by 57 % in 36-week-old rats), an increase in the number of sinusoidal capillaries in the liver, a significant decrease in the levels of serum alkaline phosphatase, AST and ALT. In 36-week-old rats, normalization of total and direct bilirubin was observed. Conclusions . The promise of an integrated approach for the treatment of toxic liver cirrhosis in rats of different age groups has been demonstrated: polytherapy prevents the development of the inflammatory reaction, reduces the area of connective tissue and damage in the liver, helps normalize biochemical parameters of blood serum, while in 36-week old rats the effects of the drugs were more pronounced in comparison with 12-week-old animals.

The liver has a big potential for self-healing, but the activity of regeneration decreases with age. Changes are occurring, including in the functional activity of various liver cell populations, the study of the characteristics of which can become the basis for the development of new therapeutic approaches to the liver diseases treatment at older people. The aim of this research was to study the level of stem and progenitor cells and the functional activity of the healthy liver from age- different rats. Material and methods. Experiments were carried out on Wistar rats aged 6 and 12 months. Ultrasound and histological examination of the liver from rats was used to assess morphological changes. The lipid profile of blood serum was evaluated by biochemical methods. Cytometric methods were used to study the surface and intracellular antigens of stem and progenitor cells isolated from the bone marrow, arterial blood and liver of rats. Results and discussion. In 12-month-old male Wistar rats, compared with 6-month-old rats, excessive formation of extracellular matrix components, disruption of tissue architecture, development of portal hypertension, as well as an increase in the concentration of cholesterol, triglycerides, high- and low-density lipoproteins were revealed. We identified age- related differences in the content of hematopoietic and mesenchymal stem cells, epithelial cells (CD45–CD326+) in the bone marrow, blood and liver of rats. In the liver parenchyma, the populations of hepatocyte precursors (CD45– CD326+CD133+), oval cells (CD45–CD326+CD133+CD90+). At the same time, the level of all cell populations in the liver parenchyma of rats expressing the intracellular marker Sox9 was higher in one-year-old animals compared to younger ones, regardless of the cell phenotype. Conclusions. In the liver of 12-month-old rats, compared to 6-month-old rats, the number of cells expressing Sox9, lymphocytes with an inflammatory phenotype increases, the number of stem cells and various populations of epithelial and endothelial cells decreases, which leads to a decrease in the regenerative capacity of the liver, disruption of the tissue architecture of the organ and changes in lipid metabolism. These changes largely determine the increased susceptibility with age to the development of chronic liver diseases.

The enhancement of early diagnosis of lung cancer is mandatory to improve prognosis. We observed the increase cancer stem cells (CSC) and circulating tumor cells (CTC) in blood of mice on d3 after Lewis lung carcinoma (LLC) cell implantation before disease symptom. Besides we have developed an approach to reprogramming immune cells by inhibiting the MAPK/ERK pathway through MEKi and the PD-1/PD-L1 immune checkpoint signaling pathway. We hypothesized that reprogramming of spleen CD8+ T-cells could also create a population of immune cells with high antitumor activity. We reprogrammed CD8+ T-cells derived from the spleen of C57BL/6 mice (rsCD8+T-cells). The rsCD8+T-cells were able to migrate to lung and were more resistant after the exhaustion in vitro in comparison with naïve sCD8+T-cells. In the orthotopic LLC model, the rsCD8+T-cells therapy increased the amount of proliferating CD8+ and CD4+ T-cells in blood and lung from mice. The amount of CSC decreased in the blood and lung of mice treated with rsCD8+ T-cells. A morphological study revealed a decrease in the number of metastases in lung tissue. The antitumor effects of rsCD8+T-cells are based on the activation of the host immune response by increasing the populations of CD8+ and CD4+ T-cells and apoptosis of CSCs.

Idiopathic pulmonary fibrosis (IPF) is one of the most common forms of interstitial lung disease, characterized by progressive parenchymal fibrosis and respiratory failure. In a model of bleomycin-induced pulmonary fibrosis, the antifibrotic and anti-inflammatory activity of Longidaze (Bovhyaluronidase Azoxymer), which contains a conjugate of the hyaluronidase enzyme with a high molecular weight synthetic carrier azoxymer bromide, was investigated. Experiments were conducted in male C57BL/6 mice. Longidaze was administered at different doses by intranasal and intramuscular routes. Histology, hematology, and enzyme-linked immunosorbent assay were used in the study. The use of Longidaze reduced pulmonary fibrosis, as evidenced by an improvement in histopathologic damage to the lungs, a decrease in the area of connective tissue, and the levels of profibrotic factors (TGF-β1, hydroxyproline, collagen I) in lung tissue. In addition, Longidaze inhibited the inflammatory response in pulmonary fibrosis, and decreased the levels of IL-6, TNF-α, and hyaluronic acid in lung tissue and the recruitment of inflammatory cells into lung tissue. The highest therapeutic efficacy was observed with the use of Longidaze at doses of 120 and 1200 U/kg intramuscularly, which was superior to that of the reference drug pirfenidone axunio. The data presented in this study suggest that Longidaze is a new and promising drug for the treatment of IPF that warrants further investigation in patients with fibrotic interstitial lung disease.

Idiopathic pulmonary fibrosis (IPF) is one of the most common forms of interstitial lung disease characterized by progressive parenchymal fibrosis and respiratory failure. In a model of bleomycin-induced pulmonary fibrosis, the antifibrotic and anti-inflammatory activity of Longidaza (bovhyaluronidase azoxymer), which contains a conjugate of the hyaluronidase enzyme with a high molecular weight synthetic carrier azoxymer bromide, was investigated. Experiments were conducted in male C57BL/6 mice. Longidaza was administered at different doses by intranasal and intramuscular routes. Histology, hematology and enzyme-linked immunosorbent assay were used in the study. The use of Longidaza reduced pulmonary fibrosis as evidenced by an improvement in histopathologic damage to the lungs, a decrease in the area of connective tissue and the levels of profibrotic factors (TGF-β1, hydroxyproline, collagen I) in lung tissue. In addition, Longidaza inhibited the inflammatory response in pulmonary fibrosis, decreased the levels of IL-6, TNF-α, hyaluronic acid in lung tissue and the recruitment of inflammatory cells into lung tissue. The highest therapeutic efficacy was observed with the use of Longidaza at doses of 120 and 1200 U/kg intramuscularly, which was superior to that of the reference drug pirfenidone axunio. The data presented in this study suggest that Longidaza is a new and promising drug for the treatment of IPF that warrants further investigation in patients with fibrotic interstitial lung disease.

We have developed an approach to reprogramming immune cells by inhibiting the MAPK/ERK pathway through MEKi and the PD-1/PD-L1 immune checkpoint signaling pathway. We hypothesized that reprogramming of spleen CD8⁺ T-cells could also create a population of immune cells with high antitumor activity. We reprogrammed CD8⁺ T-cells derived from the spleen of C57BL/6 mice (rsCD8⁺T-cells). In the orthotopic LLC model, cell therapy with rsCD8⁺T-cells increased the amount of proliferating CD8⁺ and CD4⁺ T-cells in blood and lung tissue from mice. The amount of cancer stem cells (CSC) decreased in the blood and lung of mice treated with rsCD8⁺ T-cells. A morphological study revealed a decrease in the number of metastases in lung tissue. The antitumor effects of rsCD8⁺T-cells are based on the activation of the host immune response by increasing the populations of CD8⁺ and CD4⁺ T-cells and apoptosis of CSCs.

Using a model of Lewis lung carcinoma (LLC) in vitro and in vivo, we previously demonstrated increased antitumor activity in CD8⁺ T-cells reprogrammed with an MEK inhibitor and PD-1 blocker. In this follow-up study, we carried out the reprogramming of human CD8⁺ T-cells (hrT-cell) using the MEK inhibitor and PD-1 blocker and targeted LLC cells. The effects of hrT-cell therapy were studied in a mouse model of spontaneous metastasis of a solid LLC tumor. We found antimetastatic activity of hrT-cells, a decrease in the number of cancer cells and cancer stem cells in the lungs, and an increase in the number of T-cells in the blood (including effector T-cells). Thus, reprogramming of human CD8⁺ T-cells with an MEK inhibitor and PD-1 blocker with targeted training by tumor target cells is a potential platform for developing a new approach to targeted lung cancer therapy.

Current methods for diagnosis and treatment of small cell lung cancer (SCLC) have only a modest efficacy. In this pilot study, we analyzed circulating tumor cells (CTCs) and cancer stem cells (CSCs) in patients with SCLC to search for new diagnostic and prognostic markers and novel approaches to improve the treatment of the disease. In other forms of lung cancer, we showed a heterogeneity of blood CTCs and CSCs populations, as well as changes in other cell populations (ALDH⁺, CD87⁺CD276⁺, and EGF⁺Axl⁺) in smokers. A number of CTCs and CSCs in patients with SCLC have been shown to be resistant to chemotherapy (CT). High cytotoxic activity and resistance to apoptosis of reprogrammed CD3⁺CD8⁺ T-lymphocytes (rTcells) in relation to naive CD3⁺CD8⁺ T-lymphocytes was demonstrated in a smoking patient with SCLC (Patient G) in vitro. The target for rTcells was patient G’s blood CSCs. Reprogramming of CD3⁺CD8⁺ T-lymphocytes was carried out with the MEK1/2 inhibitor and PD-1/PD-L1 pathway blocker nivolumab. The training procedure was performed with a suspension of dead CTCs and CSCs obtained from patient’s G blood. The presented data show a new avenue for personalized SCLC diagnosis and targeted improvement of chemotherapy based on the use of both CTCs and CSCs.

CD8⁺ T-lymphocytes play a key role in antitumor immune response. Patients with lung cancer often suffer from T-lymphocyte dysfunction and low T-cell counts. The exhaustion of effector T-lymphocytes largely limits the effectiveness of therapy. In this study, reprogrammed T-lymphocytes used MEK inhibitors and PD-1 blockers to increase their antitumor activity. Antitumor effects of reprogrammed T-lymphocytes were shown in vitro and in vivo in the Lewis lung carcinoma model. The population of T- lymphocytes with persistent expression of CCR7 was formed as a result of reprogramming. Reprogrammed T-lymphocytes were resistant to apoptosis and characterized by high cytotoxicity against Lewis lung carcinoma (LLC) cells in vitro. Administration of reprogrammed T-lymphocytes to C57BL/6 mice with LLC reduced the number of lung metastases. The antitumor effect resulted from the elimination of tumor cells and cancer stem cells, and the effect of therapy on cytotoxic T-lymphocyte counts. Thus, reprogramming of T-lymphocytes using MEK inhibitors is a promising approach for targeted therapy of lung cancer.