This project aimed to investigate the cellular and molecular aspects of the biology and function of multipotent adult stem cells, specifically hematopoietic stem cells and mesenchymal stromal cells. At the beginning of the project period, mesenchymal stromal cells were isolated and characterized (phenotypically and functionally) from various tissues of human and animal origin, including bone marrow, adipose tissue, umbilical cord, dental pulp, and periodontal ligament. In the following phase, the role and mode of action of various factors, primarily the inflammatory microenvironment, on the functional properties of adult stem cells were investigated, as well as the interaction of mesenchymal stromal cells with hematopoietic, immune, and tumor cells, with a particular focus on their role in immunoregulation and tumorigenesis. This project resulted in over 60 publications and presentations at scientific conferences (including 35 in JCI-indexed journals).

This interdisciplinary project aimed to develop a new system of cold atmospheric plasma for use in biomedicine, environmental control, and nanotechnology. It consisted of four sub-projects: 1) Atmospheric discharges, 2) Plasma application in biology and medicine, 3) Plasma application in positron diagnostics, nanotechnology, and material functionalization, and 4) Measurements of biogenic volatile organic compounds. Institute for Medical Research participated in the second sub-project, led by Professor Pavle Milenković. Within this sub-project, the impact of cold atmospheric plasma on the regenerative properties of human dental-derived mesenchymal stromal/stem cells was investigated, both in vitro and in vivo (the rabbit calvarial critical-size defect model). The result of this sub-project was the publication of several papers in prestigious scientific journals and presentations at numerous international conferences.

In order to discover the mechanism underlying frequent thrombosis in hematologic malignancies, it will be initiated the inflammation dependent activation and interplay of endothelial cells, platelets, and leukocytes with subsequent formation of microparticles and nitric oxide (NO) linked to thrombus generation by induction of coagulation and fibrinolysis. Cancer-associated thrombosis (CAT) is the second leading cause of death in cancer patients including both venous and arterial thromboembolism. While CAT can be treated with anticoagulants, benefits of therapy must be balanced with the increased bleeding risks. The coagulation related cells and biomarkers will be analyzed and selected by flow cytometry from the following hematologic malignancies: multiple myeloma, lymphoma, acute myeloid leukemia, and transplanted patients with graft-versus-host disease (GvHD). Besides clinical follow-up of the patients, extensive preclinical studies will be performed using in vitro cell cultures (hypoxia chamber, RT-qPCR, immunoassays), in vivo mouse models (NO analyzer), and in silico profiling. Activation of the coagulation factors will be observed through the prothrombotic properties of endothelium (microchip flow-chamber system), neutrophil extracellular traps (NETs, spectrophotometry) and anticancer treatments. The proposed study will show inflammatory stimulation of hypercoagulability, chemotaxis of immune response, and NET formation as well as modulation of fibrinolytic system and miRNA regulated platelet activation. In addition, the equilibrium between NO production and oxygen tension will largely influence the inflammation-dependent thrombus formation, with a prognostic significance of hypoxia related gene signature. Understanding the principal mechanisms may allow the development of new therapies to safely prevent CAT in the hematologic malignancies and GvHD, while determination of the specific biomarkers in each malignancy can be used as predictors of thrombosis.

Severe acute coronavirus virus syndrome 2 (SARS-CoV-2)-induced hyperinflammation is a major reason for severe clinical outcome of coronavirus disease 19 (COVID-19), principally in male patients. Cell entry of SARS-CoV-2 depends on binding to angiotensin-converting enzyme 2 (ACE2) and androgen-regulated transmembrane serine protease 2 (TMPRSS2). We propose a novel androgen-dependent mechanism of ACE2 activation by SARS-CoV-2 in endothelial and immune cells; as well as endothelial dysfunction and hyperinflammation stimulated immune response predispose to thrombus formation in COVID-19. SARS-CoV-2 will be isolated, in vitro expanded, and used to monitor cell signaling and cytokine storm by immune assays and viral load by RT-qPCR. Related androgen pathways will be pharmacologically targeted. Immune-endothelial cell co-cultures will be performed for thrombosis development. We expect androgen-dependent SARS-CoV-2 host cell infection and mediated inflammation in thrombotic complications. This translational research will reveal androgen pathways implication in SARS-CoV-2 infection and therapeutic strategies to speed up patient recovery.

Project collaborators: Dr. Aleksandra Nikolić, Dr. Branko Bobić, Dr. Tijana Štajner, Dr. Ivana Klun, Dr. Jelena Srbljanović, Dr. Vitomir Đokić, Dr. Vladimir Ivović, Dr. Marija Vujanić, Dr. Ksenija Slavić, Dr. Irena Rajnpreht, Dr. Aleksandra Uzelac, Vladimir Ćirković, Milena Stopić, Dr. Neda Bauman, Olivera Lijeskić

The aim of this multidisciplinary project, which integrates basic and applied research on infections caused by leading Apicomplexa pathogens, is to improve the control of toxoplasmosis and malaria by improving therapeutic options and developing strategies for the prevention of infection, which will contribute to the improvement of control both at the clinical level and at the level of public health. By mobilizing a critical mass of human resources (17 PhDs, including two foreign scientists who are leading in their fields of expertise, and 7 PhD students) and material resources (8 academic institutions, with which significant equipment was acquired for the project mainly in the last few years), the project will carry out a significant volume of research, the expected results of which are numerous and diverse and include the range from academic (scientific papers and doctoral theses), results directly applicable in the clinic as well as in the field of public health, to the potential for the development of new drugs. The scientific importance of the planned results lies in the contribution to the knowledge of the pathogenesis of malaria and toxoplasmosis. Basic research on the metabolic processes and transport molecules involved in these infections contributes to the understanding of the complex relationship established between the parasite and the host, and indicates potential sites of action of future drugs. The development of new transgenic lines as well as their characterization will increase the arsenal of tools available for the identification of further therapeutics. The synthesis of new active substances and the eventual identification of those with activity against P. falciparum and T. gondii will represent in itself a contribution to the repertoire of chemotherapeutics, with enormous development potential.

Applied research has immediate clinical significance. The introduction of new diagnostic methods, such as molecular diagnostics of malaria, will represent a contribution to the clinical care of these patients in Serbia. Also, in the same sense, knowledge of the genetic variability of T. gondii and P. falciparum strains that are isolated in Serbia will be useful because it will contribute to treatment with a more adequate choice of drugs, while it will also have a wider significance because they contribute to knowledge of the molecular map of these organisms at the level of the region, Europe and the world. At the end, based on the knowledge of the epidemiological parameters of toxoplasmosis in Serbia, including local risk factors, and insight into the incidence of congenital toxoplasmosis in Serbia, as a result of the project, a strategy of the national prevention program as well as a Good Practice Guide for the clinical management of toxoplasmosis will be developed. The obtained epizootiological data will indicate ways to reduce animal reservoirs of toxoplasmosis. In addition to the aforementioned, the expected results of the project include seven planned doctoral dissertations, the number of which indicates the size of the scope of the research, as well as the role that the applicant and the entire research team of the project play in the education of young researchers.