National Science Centre

The aim of this project is to test how sildenafil modulates the immune response of the future mother. As part of the project, research is being carried out on the effect of sildenafil on induced temporal NK cells (idNK), NKT cells, Th1 / Th2 / Th17 cells and regulatory lymphocytes - Treg. Drugs that reduce the activity of NK cells or inflammatory factors, as well as immunomodulators that do not have a teratogenic effect on the fetus, are the future of therapy for recurrent abortions caused by the immune system. We are constantly looking for substances that reduce the activity of NK cells in peripheral blood. Expected results: The elucidation of the mechanism responsible for the beneficial effects of sildenafil in the treatment of habitual miscarriages may, in the future, enable the widespread use of sildenafil in therapy, and may also help to identify other more effective drugs in this group of patients. The above-described studies may also help to better define the target group of patients qualified for sildenafil treatment.

The aim of the project is to investigate the possibility of using the enriched reality techniques during liver surgery, allowing to minimize their duration, improve the patient's safety and increase the efficiency in navigation and spatial orientation activities. As a result of the project, the research provided for in the project will allow better use of imaging techniques in cooperation with the surgeon by: using the natural mechanism of human vision (eye observation) without the need to adapt to artificial stereoscopic visualization or the need to change the observation point from the patient to monitors by projecting on the surface of the examined organ its internal structures. This type of approach will allow to increase the efficiency of surgeons and will contribute to minimizing the time of procedures, reducing the number of perioperative and postoperative complications and increasing the surgical effectiveness.

Bacteriophages (phages) are viruses specific to bacterial cells. Until now, the main application of bacteriophages in medicine has been the treatment of bacterial infections. However, there are also data in the literature indicating the possibility of inhibition of pathogenic virus infections by phage virions. The aim of the project is to evaluate the impact of the highly bacteriophages T4 (E. coli phage) and A5 / 80 (staphylococcal phage) and their dsDNA on the main stages of the human adenovirus type 5 (HAdV-5) replication cycle, including adsorption, viral early and late protein synthesis , intracellular transport, assembling of virions and their release from cells. It is also planned to evaluate the potential antiviral activity of dsDNA of both tested bacteriophages. In addition, the influence of phages on the expression of TLR genes (and proteins involved in signal transduction from these receptors), as well as genes encoding HAdV receptors (CAR, αv and β3 integrin chains) will be investigated - induction of changes in the expression of these genes is another possible activity of phages, which may be responsible for their antiviral activity. The influence of both phages and their dsDNA on the production of type I interferon as a result of stimulation of these receptors will also be investigated. EXPECTED RESULTS: For the first time, the effect of highly purified bacteriophage virions (and their dsDNA) on the major stages of the pathogenic virus replication cycle will be thoroughly investigated. Moreover, the ability of phages to activate TLR receptors will be assessed for the first time, and their influence on the expression of the genes of these receptors and genes encoding the receptors for the pathogenic virus. As bacteriophages are the most abundant and highly diverse group of viruses, the project results could be the starting point for future extensive research into the interactions between phage and pathogenic viruses. Demonstration of the antiviral activity of phages could indicate a new and unexpected role of endogenous phages, which are a quantitatively significant component of the microflora of the human body and various animals, as a factor also involved in the antiviral resistance of the organism. While the project is a basic research project with no immediate practical application, its results could lead to the use of bacteriophages as new antiviral drugs in the future.

The aim of the project is to investigate the role of the glutamatergic system in the treatment of depression - to verify the hypothesis that blocking the NMDA receptor with magnesium, zinc or acamprosate will intensify and accelerate the antidepressant effect of fluoxetine. Animal studies and clinical trials increasingly support the role of the NMDA receptor complex in the treatment of depression. Much research has shown that antidepressants (LPDs) can block the NMDA receptor and cause adaptive changes. The concept of the glutamatergic depression theory is also associated with the involvement of brain-derived neurotrophic factor (BDNF). The data contained in the current literature show that during the use of LPD, the level of BDNF in the blood increases. BDNF has been shown to reduce mRNA expression of NMDA receptor subunits. A lot of data also indicate the participation of inflammatory processes in the pathogenesis and treatment of depression. Under the influence of LPD, a decrease in the concentration of pro-inflammatory cytokines, in particular IL-6, was observed. In studies conducted on an animal model at the Institute of Pharmacology of the Polish Academy of Sciences in Krakow, it was shown that magnesium, zinc and acamprosate, by modulating the glutamatergic system, may have antidepressant effects. The data contained in the literature concern tests performed mainly on animals, clinical studies are scarce - they concern attempts to supplement the LPD therapy with zinc and magnesium ions, there are no studies on the potentialisation of LPD with acamprosate. Research method The serum will be collected from patients who have been diagnosed with a depressive episode in the course of recurrent depressive disorders according to ICD-10 or major depression according to DSM-IV. The NMDA receptor blocking effect will be assessed in three study groups: I - fluoxetine + magnesium; II - (Ą O fluoxetine + zinc; 111 - fluoxetine + acamprosate. Control will be fluoxetine + placebo. Serum will be drawn at 6 time points, duration of the study will be 8 weeks. The effect of NMDA receptor blocking will be assessed by a neurophysiological study: -assessment of changes in the pharmacoelectroencephalographic record (method developed at the Department of Psychiatry, Medical University of Warsaw); therapeutic response markers: BDNF and IL-6 levels, fluoxetine, magnesium, zinc and acamprosate levels of mental state: clinical status will be assessed at each time point using scales : Hamilton Depression Scale, Hamilton Scale Anxiety Scale, General Clinical Impression Scale and Adverse Symptoms Scale. The result of the project will be to develop the mechanism of action of NMDA receptor antagonists in the treatment of depression. on animals. It is planned to present the results in the form of a publication, two doctoral theses and a habilitation thesis.

Hymenolepis diminuta is the most important model species used in the study of many aspects of tapeworm infestation and parasite-host interactions. Analyzing the complex parasite-host relationships, adaptation and evolution of tapeworms requires a thorough understanding of the molecular underpinnings of these processes. Understanding the diminut genome will therefore contribute to a better understanding of the mechanisms that ensure tapeworms' evolutionary success and survival. These results will be compared with the recently available genomes of pathogenic species of the Tampidac family (Echinococcus granulosus, E. multilocutlaris, Taenia sol and um). This will allow understanding of the mechanisms responsible for the varied pathogenicity of tapeworms. An additional goal of the project will be to compare the U. diminuta inbred strain (inbreeding for over 100 generations) with the wild strain isolated from rat and human and to trace the changes in the genome that occurred during this time in the isolated tapeworm population in comparison with wild strains (human and animal) . This may be crucial in understanding the speciation process in isolated populations, which makes this research unique worldwide.

Objective and expected results: The aim of the project is to find, among plants from the Oleaceae family, compounds with potential activity promoting inflammation suppression by inhibiting neutrophil infiltration, inhibiting the production of pro-inflammatory cytokines, stimulating phagocytosis of apoptotic neutrophils and inducing the secretion of anti-inflammatory factors by macrophages. The well-documented traditional use of plants from the Oleaceae family in the treatment of inflammatory diseases, combined with their photochemical diversity, allows for a hypothesis that these plants can be a rich source of interesting new compounds with multidirectional anti-inflammatory effects. In order to verify the hypothesis, comprehensive photochemical tests will be carried out, combined with the isolation of biologically active compounds. A thorough characterization of the biological activity of the isolated compounds in vitro and in vivo will allow to determine their anti-inflammatory mechanism at the molecular level. Research method The planned research will concern 32 extracts (water, 60% ethanol, dichloromethane (DCM)) obtained from various parts of plants belonging to three genera of the Oleaceae family: Fraxinus (1 species), Syringa (1 species) and Forsythia (3 species). The work plan was divided into two parts: photochemical studies and biological studies. The main task of the phytochemical part will be to analyze the obtained extracts and fractions using the HPLC-DAD-MS / MS method. The most active extracts will be divided into fractions of different polarity. From the most active extracts / fractions, the compounds responsible for the observed activity will be isolated using the 'bio-guided isolation' method. The study of biological activity will consist of the following steps: - Preliminary evaluation of the anti-inflammatory activity of the extracts will be established based on the examination of the inhibition of oxygen burst, the release of proteases (elastase and MMP-9) and cytokines (IL-8 and ILip) in the model of human neutrophils as well as influence on the production of pro-inflammatory cytokines (IL-6 and TNF-a) and anti-inflammatory IL-10 as well as phagocytosis of apoptotic neutrophils by macrophages obtained from the THP-1 cell line. Assessment of cytotoxicity of extracts on both cell models. - For selected extracts / fractions with outstanding activity, tests will be carried out using a wider range of concentrations. Additionally, the effect on the surface expression of adhesion molecules and apoptosis of neutrophils will be determined. More in-depth studies of the effects on the production of anti-inflammatory factors (TGF-P) as well as efferocytosis and phagocytosis will be carried out on macrophages obtained from the THP-1 cell line. - In order to carry out 'bio-guided isolation', an individual model will be selected for each fraction, in which the strongest or outstanding activity was observed in the previous stages. - The isolated active compounds will be comprehensively tested on other models, which will allow to indicate their selectivity or pleiotropic effect. - The molecular mechanism of action of selected single active compounds will be determined by the impact on the NFKB, JAK / STAT, MAPK / ERK and IP3 pathways and the expression of mRNA encoding previously determined cytokines (RT-PCR, qRT-PCR) - For selected promising compounds (1-3 ) the influence on the activation state of the immune system caused by arterial hypertension in an animal experimental model will be investigated. Effect of the results Plant extracts containing secondary metabolites, which are a group of compounds with a very diverse chemical structure, and therefore generating a large number of pharmacophores with a unique spatial structure, have long been a source of new drugs. In addition, compounds of natural origin, unlike synthetic molecules, due to their biochemical functions in the plant, offer a great chance for potential interaction with proteins and the ability to penetrate intercellularly. Currently, due to the rapid development of phytochemical methods, metabolomics, biological models and rationalization of the selection of plant material, there is a renaissance of interest in a natural source in the search for new drugs. This project is in line with the trend of modern pharmacognosy / pharmacy and will allow the selection of compounds with selective or pleiotropic action that can reduce the symptoms of chronic inflammation.

The intestinal epithelial cells constitute the single layer of epithelium that forms a barrier that separates the intestinal lumen from the immunocompetent cells of the GALT system. The intestinal epithelium is now considered an important part of the gut immune system necessary for the induction and regulation of both non-specific and specific immune responses in the intestinal mucosa. Impaired immune function of epithelial cells may contribute to the development of inflammatory bowel disease (IBD) and food allergies. The main goal of the project is to evaluate the effects of bacteriophages (bacterial viruses) on the immune functions of the intestinal epithelium in vitro. Research method / methodology used All experiments will be performed on the Caco-2 cell line. In the first stage of the study, the influence of bacteriophages on the expression of selected genes important for the immune functions of the intestinal epithelium will be assessed. Gene expression will be assessed using the RT2 Profiler PCR Assay kit. Significant bacteriophage-induced changes in gene expression will be verified by measuring the production of the corresponding proteins by ELISA (for soluble proteins) or by flow cytometry (for membrane proteins). The influence of the expected results on the development of science, civilization and society. According to our knowledge, this is the first study to assess the influence of bacteriophages on the immune functions of the intestinal epithelium. The implementation of the project will broaden the knowledge of the role of the intestinal microflora in the regulation of immune homeostasis in the intestinal mucosa and the pathogenesis of IBD and food allergies.

The applicant's current research is assessing the possibility of differentiation of non-haemalopoietic stem cells obtained from mouse bone marrow (Sca-1 + CD45-CXCR4 + SSEA1 +) into lung stem cells - pncumocytes II order (AT2). The possibility of such differentiation has already been proven (Kassmeret al., 2013), however, research is currently conducted on the model of induced lung injury. The animal model is SPC-KO/SPC-TK double transgenic mice lacking the expression of a surfactant protein C (SPC-KO) and containing an apoplosis-inducing gene linked to the promoter of the SPC gene (SPC-TK). The aim of the activity to be performed is to assess the physiological ability to proliferate and self-renew second order pneumocytes and bronchioalveolar stem cells (BASC) generated in response to lung damage. To achieve the intended purpose, the applicant will isolate AT2 and BASC cells from properly prepared lungs from two groups of SPC-KO SPC-TK mice. (experimental group subjected to ganciclovir and bleomycin induced lung injury, m = 10; control group with normal lung, n = 10). Organoid cultures will be established from both cell lines, harvested after 16 days, and then some will be fixed in the form of microscopic preparations, and the other part, after disintegration of the three-dimensional structure of the organoids, will be subjected to cytometric analysis for quantitative and qualitative analysis. The results of these analyzes will be compared between the groups and will be statistically analyzed. This particular animal model was designed for the planned research due to the fact that the lack of SPC protein, which is one of the main components of the surfactant, is the cause of many human diseases (Hong et al., 2017). In addition, the use of SPC-KO/SPC-TK mice enables "targeted partial dcplection" of second order pneumocytes using ganciclovir. The discussed topic significantly affects the problem of lung regeneration necessary in various pathological conditions. Second order pncumocytes as well as bronchoalveolar lung stem cells constitute a very small number of lung cell populations that are directly responsible for the regeneration of this organ. AT2 and BASC cells are susceptible to damage, which causes slow and often insufficient lung regeneration. Therefore, there is a need to develop an effective method of their isolation from damaged lungs, multiplication and their extracorporeal differentiation, and then implantation into the damaged organ. The results obtained as a result of the planned preliminary research will become a significant contribution to undertaking a much wider project, which will be a continuation of the undertaken topic. As reported in the literature, the proliferation potential and the ability of stem cells to differentiate into cells of a different type increases significantly in damaged organs. The planned research assumes that both second-order pneumocytes and bronchoalveolar stem cells isolated from damaged lungs of SPC-KO/SPC-TK mice will be much more active, which will translate into an increased amount of organoids grown in the experimental group. The tc organoids should have a higher total number of cells than in the control group. Depending on which cell line they arise from, they should contain a higher percentage of self-renewing stem cells (in the case of the AT2 cell line), and in the case of the BASC cell line a higher percentage of both self-renewed BASC cells and second-order pneumocytes. The tc results could support the hypothesis that lung injury stimulates second order pnemocytes and bronchoalveolar stem cells to increase their physiological ability to proliferate and self-renew. Obtaining the results confirming this hypothesis will directly contribute to the attempt of extracorporeal proliferation of lung stem cells, which may be used in the future to increase the regenerative potential of this organ.

Infection with the hepatitis C virus (HCV), causing hepatitis C (hepatitis C), affects approximately 170 million people worldwide. It is an important medical problem as it can lead to serious consequences such as liver failure and hepatocellular carcinoma. The hidden course of the infection, limited access to modern treatment, and the lack of a vaccine are the reasons for the constant spread of the virus around the world. In addition, co-infection with human immunodeficiency virus (HIV-1) and HCV is increasingly observed, worsening patient survival and the effectiveness of HCV antiviral therapy. The acquired immune response associated with cytotoxic T cell activity plays a very important role in the sequelae of hepatitis C virus (HCV) infection. Approximately 20-50% of the infected eliminate the infection spontaneously due to a strong, multispecific response of these cells. In contrast, chronic hepatitis C has shown a weak T-cell response to the virus of functional "exhaustion". There is a deterioration in the secretion of inflammatory factors (IFN-γ, TNF-α and IL-2), the ability to destroy infected cells and the ability to multiply T cells after the diagnosis of the virus. These disorders, caused by chronic stimulation of the immune system, progress with the duration of the infection and are characterized by an excess of specific PD-1 and Tim-3 receptors on the surface of T cells, including those specific for HCV, which "inhibit" the ability to multiply, destroy infected cells and the secretion of inflammatory factors. Moreover, there is an increased secretion of anti-inflammatory cytokines (mainly IL-10). So far, studies on T-cell "depletion" in HCV infection have relied on the mechanisms, degree of reversibility and impact of this phenomenon on the development of chronic infection, while no other sequelae are known. In particular, whether there a relationship between T cell "depletion" and the level of diversity of immunogenic protein fragments (epitopes) of the virus recognized by these cells. In addition, how antiviral therapy for HCV infection modifies T cell "depletion" The aim of this project is to determine: 1. the relationship between T cell "depletion" and the degree of heterogeneity of immunogenic fragments of hepatitis C virus proteins recognized by T cells in patients with infection HCV and with HIV-1 / HCV co-infection; 2. how treatment of chronic hepatitis C affects the degree of "depletion" of T cells in patients with HCV infection and HIV-1 / HCV co-infection.

The results of the study conducted on a large group of patients will allow for the first time to clearly identify SNPs related to the predisposition to the development of GD in the Polish population with OR and to compare the obtained results with the results of studies in other populations (British, Chinese), to identify SNPs associated with the occurrence of specific, well-defined characterized GD phenotypes.