This disertation thesis aims to determine the effect of probiotic on the cells of immune system – human, procine and rat neutrophils and macrophages. Immune cells were exposed to 3 probiotics – Bifidobacterium bifidum, Lactobacillus rhamnosus and Enterococcus faecium, which are normally included in GIT. The first aim of this study was choose the most appropriate method for the isolation of human and porcine neutrophils and PBMC from peripheral blood. Other aims were to find out whether interactions of probiotics with neutrophils and macrophages affect their lifetime and lead to their structural changes. And whether intraperitoneal administration of probiotics lead to pro-inflammatory polarizoation of macrophages and affect viability of intraperitoneal immune cells. Structural changes of cells were detected by light and transmission electron microscopy. For detection of early and late apoptosis was used flow cytometry and for detection od cytolysis was used LDH aktivity. To determine of effect of probiotics on polarization of rat macrophages, qRT PCR was used. The results of this thesis suggest that all tested probiotics led to a significant increase in early apoptosis in porcine neutrophils and late apoptosis and cytolysis (LDH) in human neutrophils in vitro.

The main goal of this thesis was to optimize the current therapeutic approach using TLR agonists and anchored agonists of phagocytic receptor to treating cancer. The study is focused on searching a suitable agonist of TLR as the replacement of LPS.

This thesis (Application of L-carnitine to the peritoneal cavity of rat) deals with the effect of L-carnitine on the inflammatory response of the organism. L-carnitine is a common part of the body's physiological functions. It is part of the lipid metabolism in the mammalian organism. Its effect helps to transfer fatty acids from the cytosol to the mitochondria. The literary part deals with the current state of L-carnitine research and its effects in the body. It also deals with methods of applying active substances to or-ganisms and blood elements, namely leukocytes and their functions associated with inflammatory reaction. The experimental section deals with the influence of L-carnitine on the course of inflammatory reaction in the peritoneal cavity. It also deals with the determination of the dynamics of the inflammatory response, which is evalu-ated using absolute and differential leukocyte counts at two time points.

Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...

The objective of this diploma thesis was to determine the influence of the application of 5-aminosalicylic acid (5-ASA) in the peritoneal cavity on the initiation and course of the inflammatory reaction. For these purposes, the rat was chosen as the model animal on which the model of intraperitoneal lavage was applied. The rat’s peritoneal cavity is easily accessible and reflects the functions and reactions of the immune system. For the purposes of the experiment, the rats were divided into 3 groups; in the first group, the peritoneal cavity was assessed in its physiological condition, without any prior application of the substance. PBS was applied to the second group of rats, and the evaluation of the absolute numbers of cells followed after 4 and 24 hours using the Bürker counting chamber and optical microscopy. In the last group, mesalazine was applied, the exposure of which was 4 hours for one half of the group and 24 hours for the other. These time intervals were followed by the evaluation of the absolute number of cells. In order to determine the differential numbers of cells for all the samples, the coated glasses were coloured and evaluated. The greatest statistically relevant difference was identified in the case of the neutrophil population, where the neutrophil share increased from 0-5 % in the intact cavity to up to 35 % in the cavity after the PBS application. Out of all the experimental groups, the population of lymphocytes was relatively stable; the share of macrophages was, statistically, considerably lower for the groups after the mesalazine application. These results indicate that the application of PBS as an inert substance did not cause such a major reaction regarding the influx of neutrophils as the application of 5-ASA.

The aim of this thesis was study of replacement of LPS in innate immunity based cancer immunotherapy. Application of poly (I:C) with mixture of mannan-SMCC + R-848.HCl leads to synergy, higher reduction of tumor growth and significant prolongation of survival. The significance and mechanism of action of neutrophils have also been studied within this therapy.

This study examines the role of innate and adaptive immunity in the immunotherapy based on the combination of the ligands stimulating phagocytosis anchored in the tumour cells membrane and the mixture of TLR agonists. This immunotherapy is primarily focused on the innate immunity activation and induces strong inflammatory infiltration, which neutrophils and NK cells are part of. Therefore, the next aim of this study was to evaluate the anti-tumour activity of neutrophils and NK cells. For examination malignant melanoma and pancreatic adenocarcinoma mouse tumour models were used.

Immunotherapy became a very promising approach for cancer therapy. Tumor cells are eliminated using the body's own immune system with minimal negative effect on healthy tissue. This thesis is focused on immunotherapy based on activation of innate immunity, specifically on intratumoral application of ligands stimulating phagocytosis and Toll-like receptor ligands. This therapeutic approach was tested in several types of tumor mouse models, such as melanoma B16-F10, pancreatic adenocarcinoma, and pheochromocytoma. The composition of the therapeutic mixture as well as the application schedule were optimized in our studies. Subsequently the underlying mechanisms involved in tumor elimination during this therapy were investigated.

Type 1 diabetes (T1D) is an autoimmune disease with multifactorial aetiology that involves an attack of self-reactive cytotoxic CD8 lymphocytes on insulin-producing beta cells in the pancreas. In the T1D pathophysiology, both innate and adaptive immunity mechanisms cooperate in the development of inflammation leading to autoimmune destruction. Autoreactive T lymphocytes are the canonical destructors of the beta cells, and B cells produce autoantibodies; the innate immunity cells are considered the initiators of the pathological autoimmune reaction by promoting T and B cell activation. Here, we provide evidence of both innate and adaptive immunity cell types dysregulation in patients with T1D, and that these changes occur before the onset of the disease. The changes in T regulatory lymphocytes (Tregs) and B cell subpopulations occur already in asymptomatic T1D first-degree relatives. During the first year after the onset of the disease, there is a gradual decrease in the neutrophil numbers in the periphery, which probably infiltrate the pancreas. We have focused more closely on the innate immunity dysregulation and its contribution to T1D pathogenesis. Initially, we describe that neutrophil products called neutrophil extracellular traps (NETs) are able to induce IFNγ-producing T cells through...