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Proportional and functional characteristics of particular neutrophil subpopulations in umbilical cord and peripheral blood
Miková, Eliška ; Hrdý, Jiří (advisor) ; Krulová, Magdaléna (referee)
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...
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Immunologic characteristics of milk: Differences between nonallergic mothers
Hrdý, Jiří
Immunologic Characteristics of Milk: Differences Between Nonallergic and Allergic Mothers Maternal milk influences positively the development of newborn's immune system. Differences between milk of healthy and allergic mothers are not well established and the results concerning effect of allergic milk on newborn's immunity are controversial. In the present work, the effects of healthy and allergic colostrum/milk on the stimulation of cord blood lymphocytes of children of healthy and allergic mothers were studied. The properties of milk and cord blood cells were tested using cytokine and immunoglobulin detection by immunoenyzmatic methods (ELISA and ELISPOT respectively), cell cultivation, 3 H-thymidine incorporation and cytokine mRNA expression by real-time PCR. According to the results obtained, there are not larger differences between healthy and allergic milk. Colostrum/milk in high concentration suppresses proliferation of cord blood lymphocytes after stimulation by polyclonal activators. Lymphocytes of children of allergic mothers incorporate 3 H-thymidine more intensively than lymphocytes of children of healthy mothers. On the other hand, colostrum/milk increases Ig production by stimulated cord lymphocytes in vitro. Cocultivation of milk cells with stimulated cord mononuclear leucocytes in Transwell...
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Immunologic Characteristics of Cord Blood in Children with Increased Risk of Allergy Development Preventive Use of Probiotics
Hrdý, Jiří ; Prokešová, Ludmila (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Ulčová-Gallová, Zdeňka (referee)
Allergy is one of the most common diseases. Identification of early prognostic markers pointing to an increased risk of allergy development is therefore of increasing importance. Cord blood represents an easily attainable clinical material for searching for prognostic markers signalizing future allergy development. Proportions of Th1 cytokines, Th2 cytokines and regulatory cytokines were tested in cord blood of children of allergic mothers (children in relatively high risk of allergy development) in comparison with cord blood of children of healthy mothers (low risk children). Also the activities of lymphocytes, dendritic cells (DC) and regulatory cells (Tregs) were compared in children of healthy and allergic mothers. The generally increased activity of both in vitro stimulated and non-stimulated mononuclear cord blood leukocytes was proved in children of allergic mothers in comparison with low risk children. The increased activity of DC of high risk children was detectable only after polyclonal stimulation. Significantly less pronounced functional properties of cord blood Tregs were found in children of allergic mothers when compared with children of healthy mothers. The increased reactivity of lymphocytes and DC together with the decreased activity of Tregs can support an easier...
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The Role of Microbiota in the Pathogenesis of Psoriasis
Stehlíková, Zuzana ; Jirásková Zákostelská, Zuzana (advisor) ; Demnerová, Kateřina (referee) ; Hrdý, Jiří (referee)
Psoriasis is a chronic, immune-mediated inflammatory skin disease. Its pathogenesis is associated with dysregulated cooperation among keratinocytes, innate and adaptive immune cells, coupled with environmental triggers, including microbiota. The aim of our study was to describe the microbiota composition in psoriasis and explore the role of bacteria and fungi in the pathogenesis of this disease. We used a mouse model of psoriasis induced by topical application of imiquimod (IISI) in both germ-free (GF) mice and conventional (CV) mice with microbiota manipulated by administration of a mixture of broad-spectrum antibiotics (ATB). ATB treatment markedly changed the intestinal but not the skin bacterial diversity and led to higher resistance to IISI in CV mice. Metronidazole was the most effective antibiotic, alleviating IISI symptoms in CV, but not in GF mice. This confirms that the effect of metronidazole on IISI was microbiota- dependent. Additionally, we characterized the microbiota composition of psoriatic lesions and unaffected skin in psoriatic patients compared to healthy controls, as well as the impact of different sampling approaches on uncovering cutaneous microbiota composition. We observed significant differences in α- and β-diversities when comparing identical samples sequenced on V1V2...
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Maternal allergy status has no impact on neonatal immune responses to allergen stimuli
Lohonková, Adéla ; Hrdý, Jiří (advisor) ; Černý, Jan (referee)
The alarming increasing incidence of allergic diseases leads to the effort to identify the group of markers with prognostic potential pointing reliably to an allergy development at a later age. Identification of such marker or a group of markers pointing to a higher risk of developing an allergy would allow early implementation of preventive measures to block further development of the allergic disease. Umbilical cord blood appears to be the ideal biological material for the search for prognostic markers that are suitable for further study. The ability of umbilical cord blood cells to respond to stimulation by common allergens by producing cytokines according to a pattern dependent on the allergic status of the mother could be the predictive feature sought. The presented study focuses on the determination of cytokines typical for Th1- : interferon gamma (IFN-γ), Th2-: interleukin 5, interleukin 13 (IL-5, IL-13) a regulatory T cells (Tregs): interleukin 10 (IL-10) immunity responses at the level of gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) followed by determination of the protein production itself in the cell culture supernatant. Newborns were divided into two groups according to the allergic status of their mothers: children of healthy mothers (newborns with a...
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Immunoregulatory characteristics of immune cells of children of allergic and non-allergic mothers and the possibility of their modulation with probiotic E. coli strain O83:K24:H31
Černý, Viktor ; Hrdý, Jiří (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Černá, Marie (referee)
Due to high incidence, medical and socioeconomic burden and impact on individual quality of life and productivity, allergic disorders are a crucial issue for 21st century immunology. Much still remains to be elucidated, particularly regarding the very early processes in allergy development. In order to introduce timely, effective preventive measures, novel, more reliable predictive factors of allergy risk also need to be established. Dysregulation of proper balance between the branches of immune response, particularly unwarranted dominance of Th2, is the underlying cause of allergy. After birth, new immune balance needs to be established to prepare the neonate for adequate reactivity towards newly encountered environmental stimuli. Regulatory T cells (Treg) play a central role in finely setting this balance and inducing tolerance towards harmless environmental antigens, including allergens. Interactions with external factors, most importantly microbiota, modulate this process during the early postnatal "window of opportunity." Analysis of cord blood Treg of children of allergic mothers uncovered decreased presence of function-associated surface markers and lower production of IL-10. Furthermore, decreased proportion of Helios- induced Treg was observed in children with higher risk of allergy....
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The effect of progesteron administration on T regulatory cells in relation to preterm deliveries
Škvorová, Anna ; Hrdý, Jiří (advisor) ; Krulová, Magdaléna (referee)
During pregnancy, the maternal organism has to cope with the presence of foreign paternal antigens, allow and support the development of a semialogenic fetus, while maintaining its defense against pathogens. This condition is achieved by a dynamic balance of complex immuno-endocrine interactions. A number of cells with specific properties contributes to the tolerogenic setting during pregnancy. Very important role plays the population of T regulatory lymphocytes (Treg). Treg levels increase physiologically during pregnancy and their decline is associated with term and preterm labor. Premature birth is a serious socio-economic problem, affecting around 10 % of births worldwide. In the case of increased risk of premature labor, progesterone administration has been successfully used in clinical practice. One hypothesis says that progesterone induces an increase in Treg population and delays premature labor in this way. The aim of the thesis was to monitor the effect of progesterone therapy in vivo on the amount and functional properties of CD4+ CD25+ FoxP3+ Helios+/- Treg and to verify whether long-term vaginal administration of progesterone positively correlates with an increase in Treg counts. Another goal was to determine whether progesterone promotes Treg generation and the production of...
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Cytototoxic effects of antiseptics in devices for chronic wound healing
Kejzlarová, Leona ; Pavlík, Vojtěch (advisor) ; Hrdý, Jiří (referee)
Chronic wounds are often colonized by pathogens bacteria. The occurrence of infection on the surface results in an injured and delayed healing process. Therefore, the use of antimicrobial agents that eliminate microbiological stress and prevent infection is required for the treatment of chronic wounds. Microbial agents include antibiotics, but pathogens are able to develop resistance to them. Antiseptic agents are therefore used, which act non-selectively. Antiseptics may be cytotoxic to human tissue cells and cause a delay in wound healing. A certain balance needs to be struck between antimicrobial activity and cytotoxicity. The most commonly used active substances with microbicidal properties in antiseptic preparations are: silver, iodine, chlorine, octenidine, polyhexanide, and chlorhexidine. This work describes the mechanisms of action of these agents and compares the clinical and laboratory knowledge of their effectiveness in chronic wound healing and cytotoxicity against cells involved in this process. Keywords: Chronic wounds, antiseptic, cytotoxicity, octenidine, chlorhexidine, polyhexamethylen, PHMB, iodine, silver, chlor
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The role of neutrophils in preeclampsia
Miková, Eliška ; Hrdý, Jiří (advisor) ; Grobárová, Valéria (referee)
Preeclampsia is currently one of the leading causes of maternal morbidity and mortality. Disease development is caused by multiple factors but the main trigger remains unknown. Preeclamptic placentas are defined by increased inflammatory environment. One of the first cells to enter the site of inflammation are neutrophils. Current studies uncover considerable heterogeneity of neutrophils which used to be considered as terminally differentiated population with a very limited plasticity. Disbalance in the proportional and functional properties of proinflammatory and immunosuppressive neutrophil subpopulations including myeloid derived suppressor cells can lead to severe pregnancy complications such as preeclampsia. Better understanding of the origin of abnormal neutrophil subpopulations and their role in the pathogenesis of preeclampsia could help reveal possible targets for therapeutic intervention. Key words: neutrophil subpopulations, preeclampsia, NETosis, myeloid derived suppressor cells, myeloperoxidase, elastase, PD-L1, inflammation
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Immunomodulatory properties of TES
Holečková, Markéta ; Hrdý, Jiří (advisor) ; Macháček, Tomáš (referee)
Toxocara canis and Toxocara cati are parasitic roundworms. Their definite hosts are canines or felines. Human infection is also possible and in that case these parasites cause human toxocariasis. Larvae of Toxocara spp. in the host release proteins called TES (Toxocara Excretory-Secretory antigens). The main role of these proteins is to ensure long-term survival of the parasite in the body of the host. The surface of the larvae is covered by coat made of some of those proteins, which increases the parasite's chances of survival. Other proteins secreted by Toxocara spp affect production of cytokines of the infected organism and parasite modulates the immune response to infection by that. Presence of the larvae results in inhibition of immune response based on Th1 cells and promotes Th2 immune response, during which levels of IgE and eosinophiles are elevated. Production of regulatory T lymphocytes is also stimulated. Key words Toxocara spp., toxocariasis, TES, immunomodulation, regulatory T lymphocytes, cytokines
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