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Host-microbe interactions and its consequences for intestinal inflammation and carcinogenesis
Kejzlarová, Leona ; Kverka, Miloslav (advisor) ; Smrž, Daniel (referee)
A number of physiological and pathological processes, including the transition from chronic inflammation to cancer, are affected by commensal microbes. However, abundance of microbes and ability to produce active metabolites in the intestine depend on environmental factors, particularly diet. Microbes can influence this process in two ways, by producing genotoxic substances that directly damage the epithelium or by stimulating the inflammatory response. The aim of my thesis was to study the interaction among gut microbiota, diet and the immune system with the subsequent influence on the development of colorectal cancer (CRC) in an experimental mouse model. Animals were fed synthetic diets containing either normal amounts of animal protein (17%; KD) or elevated amounts of animal protein (51%; HPD) throughout the experiments. Two weeks after the diets were introduced, intestinal tumors were induced by administering azoxymethane (AOM) and inducing acute inflammation with 2% sodium dextran sulfate one week after AOM injection. At the end of the experiment I evaluated the number of tumors in the colon and the status of the immune response in the intestine, mesenteric lymph nodes and spleen. To study the effect of macrophages, a similar experiment was performed in animals with depleted macrophages using...
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The immune response of mice against avian schistosomes: from local to systemic perspective
Majer, Martin ; Macháček, Tomáš (advisor) ; Kverka, Miloslav (referee) ; Sombetzki, Martina (referee)
In the host bodies, helminths often undergo complicated migrations that afflict various organs. Defining the immune response is then usually focused only on isolated spatio-temporal fragments but lacks the whole-body context. To broaden the knowledge about the tissue-specific and systemic immune response, this thesis focused on mice infected with two model species of avian schistosomes of the genus Trichobilharzia. T. regenti penetrates the skin and enters the spinal cord within a few days. Most of the parasites die in the skin which is accompanied by an influx of leukocytes. We confirmed that it was partially mirrored also in skin draining lymph nodes by increasing the T helper 2 cells proportion. Interestingly, there were reduced numbers of T cells in the spleen with an increased proportion of the regulatory T cells. Therefore, we conclude that adaptive systemic response differed and the regulatory phenotype dominated. However, the skin phase-induced eosinophilia in blood influenced the milieu of the central nervous system (CNS). Schistosomula which entered the spinal cord triggered inflammation and enhanced expression of eotaxins, which attracted the eosinophils from the blood into the CNS. The antigen-presenting cells contribute to the communication of CNS with the periphery. We employed and...
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In vitro modulation of immune cells for adoptive cellular cancer immunotherapy
Kalkušová, Kateřina ; Smrž, Daniel (advisor) ; Kverka, Miloslav (referee)
Cancer is one of the leading causes of death in developed countries. Its traditional treatment is based on surgical removal of the tumor and metastases, radiotherapy, and chemotherapy. Recently, many new therapy options, including immunotherapy, have been investigated. Cancer immunotherapy seems to be a very promising treatment option as it has experienced many successes in the last few decades. However, there is still a number of patients not responding to today's immunotherapy methods. Adoptive cellular immunotherapy is one of those immunotherapeutic methods. This immunotherapeutic modality uses ex vivo prepared immune cells that participate in anti-tumor responses. Nowadays, most research is focused on the use of T cells, although many other cell types are considered, including dendritic cells. This thesis is focused on the modulation of dendritic cells for adoptive cellular cancer immunotherapy. The aim of the practical part is to evaluate the influence of beta2-microglobulin on the maturation of monocyte-derived dendritic cells. Key words: Adoptive cellular immunotherapy, dendritic cells, beta2-microglobulin, cancer
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Gut barrier in the pathogenesis and diagnostics of necrotizing enterocolitis and inflammatory bowel disease
Coufal, Štěpán ; Kverka, Miloslav (advisor) ; Nevoral, Jiří (referee) ; Stříž, Ilja (referee)
Disruption of gut microbiota, altered mucosal defense, inappropriate immune response and gut barrier damage are all typical features in the pathogenesis of both necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). Despite of intensive research, the exact pathogenesis of both diseases remains unclear and the diagnostics and outcome prediction are still problematic. Therefore, we analyzed the role of gut-associated and inflammatory biomarkers, with respect to different aspects of gut barrier dysfunction in the pathogenesis of both disease, with the aim to improve the diagnostics and to predict the disease course and outcome. Using ELISA, we found that patients who will later develop NEC have significantly higher intestinal fatty acid-binding protein (I-FABP) than infants who will later develop sepsis already in first hours after NEC suspicion. Urinary I-FABP had high sensitivity (81%) and specificity (100%) and its addition to currently used gold standard for NEC diagnosis increased its sensitivity and negative predictive value. We found that serum amyloid A (SAA) was the strongest factor for prediction of the most severe stage of NEC. The combination of intestinal and liver FABP with SAA predicted the length of hospitalization in NEC patients and the low level of SAA predicted short...
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Microbiota as a modulator of carcinogenesis
Benešová, Iva ; Kverka, Miloslav (advisor) ; Krulová, Magdaléna (referee)
Many studies show the ability of gut microbes to modulate the anti-tumour immune response by direct triggering the immune cells or by bacterial metabolites. Interestingly bacteria may even migrate to the tumour tissue and orchestrate the immune response on site. These anti-tumour effects can be improved by the administration of immune checkpoint inhibitors (ICI). Notably, some microbial effects occur only in the presence of ICI. On the contrary, microbiota may also promote tumour growth and negatively impact the effects of ICI therapy. We have disrupted the gut microbiota homeostasis by antibiotics (ATB) to study the effects of gut microbiota on the ICI. This disturbance led surprisingly to reduced tumour growth and enhanced pro-inflammatory immune response not only in the gut but also within the tumour tissue, where especially IFN-γ orchestrated the anti-tumour immune response. Importantly the anti-tumour immune response could be transferred through colonisation of germ-free mice by ATB-changed gut microbiota if concomitantly anti- programmed cell death protein 1 (αPD-1) monoclonal antibody was administrated. These mice had elevated levels of segmented filamentous bacteria (SFB), which induced systemic immune response with increased expression of IL-17 and elevated amounts of Th 17 cells,...
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Modulation of the Mucosal and Systemic Immunity by Microbiota in Experimental Autoimmune Uveitis
Šlemín, Johan ; Kverka, Miloslav (advisor) ; Dobeš, Jan (referee)
The use of probiotics has emerged in the last decades as a promising strategy when it comes to the treatment of inflammatory diseases. Through modulation of composition of the intestinal microbiota and the signalling it provides, probiotics can favourably tune the immune system. Beneficial effects of probiotic treatment have been documented in multiple animal inflammatory disease models. The effect of probiotic treatment on uveitis-a sight- threatening disease-has however not yet been described. In our study, we have tested two commercially available probiotics-Escherichia coli Nissle 1917 (EcN) and Escherichia coli O83:K24:H31 (EcO)-in the treatment of experimental autoimmune uveitis (EAU). The disease severity was assessed by ophthalmoscopy and histology, proportions of leukocyte populations and intracellular expression of cytokines were evaluated by flow cytometry and the gut immune environment was analysed by tissue culture and ELISA. We found that prophylactic and early oral treatment with EcN reduces the severity of EAU. However, EcO treatment does not. The effects were accompanied by immune changes including a lowered production of inflammatory cytokines in Peyer's patches, a shift in macrophage populations in ileum and mesenteric lymph nodes or a reduced IRBP-specific response of CD4+ T...
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Bacterial components in experimental intestinal inflammation prevention and therapy
Kverka, Miloslav ; Tlaskalová - Hogenová, Helena (advisor) ; Šedivá, Anna (referee) ; Stříž, Ilja (referee)
Although strong protective immune response is essential for preventing invasion by pathogens, equivalent responses against antigens originating from commensal bacteria can lead to chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Manipulating the mucosal immune responses with microbial antigens might be an excellent tool to IBD therapy or prevention. Our aim was to gain some insight into the regulation of the intestinal inflammation and to isolate bacterial immunomodulatory components that could be used in intestinal inflammation therapy and prevention. One particular mechanism of how healthy colon tissue regulates the inflammation during acute experimental colitis is through modulation of bioavailability of glucocorticoids (GCs) in gut mucosa. Here, we show that intestinal inflammation changes the local GC metabolism, which ultimately leads to decrease in inflammatory readiness of cells in the gut mucosa and in mesenteric lymph nodes. This pre-receptor regulation of GC function could represent an important homeostatic function of the gut mucosa. The actual triggers of intestinal inflammation in IBD seem to be either microbial dysbiosis or microbes with special "pathogenic" abilities, which both could be rectified by feeding with probiotics. Here, we report that oral feeding with live...
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Gut barrier in the pathogenesis and diagnostics of necrotizing enterocolitis and inflammatory bowel disease
Coufal, Štěpán ; Kverka, Miloslav (advisor) ; Nevoral, Jiří (referee) ; Stříž, Ilja (referee)
Disruption of gut microbiota, altered mucosal defense, inappropriate immune response and gut barrier damage are all typical features in the pathogenesis of both necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). Despite of intensive research, the exact pathogenesis of both diseases remains unclear and the diagnostics and outcome prediction are still problematic. Therefore, we analyzed the role of gut-associated and inflammatory biomarkers, with respect to different aspects of gut barrier dysfunction in the pathogenesis of both disease, with the aim to improve the diagnostics and to predict the disease course and outcome. Using ELISA, we found that patients who will later develop NEC have significantly higher intestinal fatty acid-binding protein (I-FABP) than infants who will later develop sepsis already in first hours after NEC suspicion. Urinary I-FABP had high sensitivity (81%) and specificity (100%) and its addition to currently used gold standard for NEC diagnosis increased its sensitivity and negative predictive value. We found that serum amyloid A (SAA) was the strongest factor for prediction of the most severe stage of NEC. The combination of intestinal and liver FABP with SAA predicted the length of hospitalization in NEC patients and the low level of SAA predicted short...
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Role of enterovirus and adenovirus infection in the pathogenesis of celiac disease
Chudá, Kateřina ; Cinek, Ondřej (advisor) ; Kverka, Miloslav (referee)
Celiac disease, a chronic immune-mediated disorder of the small intestine, manifests in a fraction of individuals with genetic predispositions consuming gluten. Environmental factors play an essential role in its triggering. The environmental stimuli may include dietary factors, infections etc. Identification of specific triggers could help in celiac disease prevention. Our research project focused on common intestinal infections in infancy. We investigated adenoviruses and enteroviruses in stool specimens of children carrying a high-risk HLA genotype for celiac disease. We aimed to determine whether these infections are associated with early markers of celiac autoimmunity, and to identify virus genotypes. To distinguish multiple infections, massive parallel amplicon sequencing was utilized. During 2001-2007, nearly 50.000 Norwegian newborns were screened within the MIDIA study for the presence of the HLA DR3-DQ2/DR4-DQ8 genotype, which is known to significantly increase the risk of celiac disease. The risk genotype was identified in 912 babies. Up to three years of children's age, monthly stool specimens were collected and archived. Blood sampling was done every three months up to the age of a year, and then annually. Periodical questionnaires on children's thrive were collected. During 2014-2016,...
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