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Influence of the citrulination of histon proteins on the expression of selected genes in myeloid cells
Tučková, Kristýna ; Dobeš,, Pavel (referee) ; Číž,, Milan (advisor)
Neutrophils are major cell type of innate immunity, that can eliminate pathogens by different mechanisms. One of these mechanisms is called NETosis, which leads to release of decondensed chromatin and citrullinated histone proteins. Citrullination is post-translational modification catalysed by peptidylarginine deiminase (PAD) and causing transformation of possitively charged arginin to neutral citrullin and can change expression of cytokine genes. Concetrations of pro-inflammatory cytokines (IL-8, TNF, IL-1) were measured after activation of PAD4 and induction of citrullination. Calcium ionophore was used to induce citrullinaton, Cl-amidine and TDFA were used as inhibitors. Production of cytokines was assessed by ELISA on protein level and by qPCR on mRNA level. It was found that induction of citrullination led to increased concentrations of IL-8 and IL-1. Elevated gene expression of IL-8 was confirmed on mRNA level. Both inhibitors were able to decrease level of histone H3 citrullination and IL-8 and IL-1 concentrations. Expression of TNF was not detected on protein and mRNA level.
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Zdravotní a produkční účinek imunomodulačního preparátu Imrestoru u vysokoužitkových dojnic
NEZNÍKOVÁ, Kateřina
This thesis deals with evaluation of the impact of immunomodulatory preparation Imrestor (Elanco Animal Health) on the health and production of high-producing dairy cows in the transition period. The experiment was done in the agricultural company Košetice in the district of Pelhřimov on dairy cows of the Holstein breed. The animals selected for the experiment were divided into two groups - control and experimental. Imrestor was applied subcutaneously to the dairy cows of the experimental group approximately one week before calving and on the day of calving. The active component of this preparation is a commercial form of a naturally occurring cytokine (bG - CSF), which specifically affects on the neutrophil precursor cells and increases their production. Blood samples were taken from all cows included in the experiment to evaluate the effect of the preparation Imrestor. At the same time was monitored their clinical health and the incidence of the most common diseases in the transition period. 81 blood samples were taken from 31 dairy cows from the control group and from 18 dairy cows from the experimental group. A biochemical and hematological examination was performed in the laboratory, where the parameters of red blood cells, trombocyte count, leukocyte count, leukocyte percentage, biochemical indicators of blood plasma and mineral content were determined. By statistical evaluation was found, that Imrestor statistically highly significantly (p <0.01) affected leukocyte counts and the percentage of neutrophil granulocytes and lymphocytes. Statistically significant differences (p <0.05) between the observed groups of dairy cows were found in the trombocyte count, percentage of monocytes and alkaline phosphatase activity.
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Development of an opsonophagocytic assay for the measurement of functional antibody activity against Bordetella pertussis
Brázdilová, Ludmila ; Bumba, Ladislav (advisor) ; Dráber, Peter (referee)
The Gram-negative pathogen bacterium Bordetella pertussis is the infectious agent causing pertussis or whooping cough. The infection is dangerous to infants, often being deadly if untreated. Since whole-cell pertussis vaccines have been replaced by acellular pertussis vaccines, pertussis has become the most prevalent vaccine-preventable disease in developed countries. Therefore, the development of a new generation of pertussis vaccines has become a high priority. Opsonophagocytic assays are one method used to assess the efficacy of new vaccines. The main objective of the thesis is to develop opsonophagocytic killing and uptake assays for the measurement of functional antibody activity against Bordetella pertussis. Neutrophils from mice and humans were isolated by three different methods and used for the assessment of different human and mouse sera in opsonophagocytic killing and uptake assays. Different experimental conditions were tested, including multiplicity of infection and serum dilutions. The opsonophagocytic uptake assay proved to discriminate between naïve and immune sera. Serum from mice vaccinated with the whole-cell pertussis vaccine enhanced opsonophagocytic uptake of B. pertussis cells into neutrophils, while serum from mice immunized with the acellular pertussis vaccine did not....
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Influence of the citrulination of histon proteins on the expression of selected genes in myeloid cells
Tučková, Kristýna ; Dobeš,, Pavel (referee) ; Číž,, Milan (advisor)
Neutrophils are major cell type of innate immunity, that can eliminate pathogens by different mechanisms. One of these mechanisms is called NETosis, which leads to release of decondensed chromatin and citrullinated histone proteins. Citrullination is post-translational modification catalysed by peptidylarginine deiminase (PAD) and causing transformation of possitively charged arginin to neutral citrullin and can change expression of cytokine genes. Concetrations of pro-inflammatory cytokines (IL-8, TNF, IL-1) were measured after activation of PAD4 and induction of citrullination. Calcium ionophore was used to induce citrullinaton, Cl-amidine and TDFA were used as inhibitors. Production of cytokines was assessed by ELISA on protein level and by qPCR on mRNA level. It was found that induction of citrullination led to increased concentrations of IL-8 and IL-1. Elevated gene expression of IL-8 was confirmed on mRNA level. Both inhibitors were able to decrease level of histone H3 citrullination and IL-8 and IL-1 concentrations. Expression of TNF was not detected on protein and mRNA level.
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Dráber, Petr (referee)
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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