Influenza is a serious illness worldwide, causing high morbidity and mortality. 10-20% of world population fall ill with influenza each year and 250 000 - 500 000 people die annually. The most efficacious protection to date is vaccination. Current vaccines are efficient only one season because of fast mutation rate of influenza virus. The effort to create an effective vaccine faces lack of potent adjuvant, which can adequately stimulate and modulate immune system to protect organism from virus infection. Moreover, todays vaccines administered parenterally do not induce immune response on mucosal surfaces. Bacillus firmus, a Gram-positive non-pathogenic bacterium, has strong immmune-modulating properties and is able to induce cross-protection when administered with influenza virus antigens. Immunization with Bacillus firmus stimulates production of neutralizing antibodies, but other mechanisms of its action remain to be elucidated. To better understand the mechanisms how is antiviral immunity enhanced by Bacillus firmus (delipidated fraction, DBF), the effect of immunization with DBF only was studied on mouse model. In last decade it has become obvious that intranasal immunization can induce both systemic and mucosal immune response and in case of influenza it can induce cross-protection. Therefore...

Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...

This work was oriented to studies of sodium and potassium dependent adenosinetriphosphatase, Na+ /K+ ATPase, which is selectively inhibited by cardioactive glycoside, ouabain. The alterations in level of this enzyme were followed in spleen lymphocytes and hippocampus prepared from rats. Detection of Na+ /K+ ATPase has been made by western blot analysis using primary antibodies oriented against α subunit of Na+ /K+ ATPase. Studies of lymphocytes were based on usage of both monoclonal and polyclonal antibodies. In studies of hippocampus monoclonal antibodies were used. The first aim of my work was to determine the alterations in the level of Na+ /K+ ATPase in spleen lymphocytes cultivated in tissue culture (i.e. under in vitro conditions) in the presence of morphine or strong mitogen, concanavalin A (ConA). The second aim of these theses was to determine the changes of Na+ /K+ ATPase α subunit in hippocampus of rats, which were under in vivo conditions exposed to stress lasting 3 days. The stress of experimental animals was induced by deprivation from sleep. The long-term incubation of spleen lymphocytes with 10 μM morphine for 48 hours did not cause a significant change of Na+ /K+ ATPase α subunit level. This result was obtain by analysis of post nuclear fraction (PNS), by use of monoclonal...

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.

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...

Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterised by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. It is a long-term process initiated months or even years prior to the clinical onset. The main role in the pathogenesis is played by T lymphocytes but other cell types are involved as well. The presence of autoantibodies in the circulation is typical even before the disease onset. Nowadays, intensive research is focused on finding individuals at risk and developing an effective prevention. During my postgraduate studies I was involved mainly in the research of T1D prediction and prevention. We investigated the relationship of established autoimmune markers - autoantibodies - and the cellular reactivity to GAD65 and IA2 autoantigens. We discovered that the reaction to autoantigens is very individual and it is influenced by the patient's autoantibody profile. These results could be relevant in planning antigen-specific immunointervention studies and improving their efficacy. We also made an attempt to improve specificity and sensitivity of a beta cell destruction marker (specifically demethylated DNA), which would enable better understanding of the beta cell decline and identification of individuals at risk of T1D development. In...

Inflammatory bowel diseases (IBD) are an autoimmune illnesses affecting gastrointestinal tract. The main types include ulcerative colitis and Crohn's disease. Recently, primary sclerosing cholangitis (PSC) has also been associated with IBD. PSC is a chronic liver disease associated with bile duct stenosis. The exact pathogenesis and etiology of these diseases is not clear, despite the great efforts of the scientific community. They are multifactorial diseases that are associated with dysbiosis of intestinal microbiota. Their diagnosis is based on for patients unpleasant endoscopic examinations and therefore the search for new serum biomarkers is needed and appreciated target of scientific interest. In the first part of diploma thesis, we focused on the reactivity of peripheral blood cells of IBD patients to 10 selected representatives of typical intestinal microbiota: Lactobacillus plantarum, Bifidobacterium adolescentis, Blautia coccoides, Roseburia intestinalis, Eubacterium rectale, Faecalibacterium prausnitzii, Ruminococcus flavefaciens, Bacteroides thetaiotaomicron, Prevotella ruminicola and Escherichia coli. Reactivity of CD, UC and PSC- IBD patients was increased after stimulation with Faecalibacterium, Lactobacillus and Prevotella. However, we got low percentage of cytokine-producing cells,...

BBSome is a protein complex crucial for trafficking of specific cargoes to the primary cilium. Although primary cilia are typically not present in cells of haematopoietic origin, such as T cells, recent research has revealed striking parallels between the primary cilium and the immunological synapse. Amongst other similarities, both structures are supposed to use the same transport machinery involving Rab8 and IFT20, the close interaction partners of BBSome. The first goal of this thesis was to investigate the role of BBSome in the biology of T cells. Using RT-qPCR, we have shown that BBSome subunits are expressed in lymphoid tissues and T cells. Studies of localization of BBSome subunits in Jurkat cell line and primary OT-I T cells revealed that the subunits have distinct localization patterns with BBS4 localizing to the centrosome and BBS1, BBS5, and BBip10 having dispersed localization. After the contact with an antigen presenting cell, BBS4 re-localizes to the immunological synapse. Mutations in BBSome encoding genes cause Bardet-Biedl syndrome (BBS), a rare ciliopathy presenting with multiorganic symptoms. The second goal of this thesis was to examine the associations between BBS and the immune system. Examination of medical records of more than 450 BBS patients revealed that autoimmune...

Introduction: Virus-specific CD8+ T cells are crucial to suppress the viral replication in HIV infection. Their functional status is important as well. Also, the chronic nonspecific immune activation of T and B lymphocytes plays an important role in the immunopathogenesis of HIV infection. Aim of the study: To analyze the frequency and functional status of HIV-specific CD8+ T cells and the expression of nonspecific activation markers on B and T cells in HIV+ patients and to assess the effect of combined antiretroviral therapy (cART) on these parameters. Patients and methods: Our cohort included 80 HIV+ patients: 36 HIV+ patients on cART, 18 patients without therapy, in whom cART was introduced during our study, 9 patients without therapy, 10 patients with primary HIV infection, 3 long-term non-progressors and 4 patients initially on cART, in whom the therapy was discontinued. Control group consisted of 34 HIV- healthy individuals. We examined CD4+ a CD8+ T cell counts, viral load, expression of nonspecific activation markers on T cells and the frequency of HIV-specific CD8+ T cells by ELISpot method and flow cytometry using MHC tetramers and intracellular cytokine detection. Results: No significant differences in HIV-specific CD8+ T cells were found between treated and untreated HIV+ patients. The frequency...