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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
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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Trapped Neutrophil Syndrome in a Border Collie Dogs
Brunclíková, Tereza ; Krejčířová, Romana (advisor) ; Chmelíková, Eva (referee)
Border Collie comes from the part of the United Kingdom which is called Border Country. It's a herding dog, which should be obedient, smart, lively, and attentive. For breeding, Border Collies must fulfil bonitation conditions. One of the mandatory tests is the test on hereditary neutropenia. Hereditary neutropenia is a disease which causes a fatal immune system failure.
Immunity; or defences; is one of the basic features for survival. Basic ability of cells of the immune system is to recognize when in contact with other molecules the structure is inherent or not. Memory, which is another ability of the immune system, ensures a prompter, more intensive, and quicker response when meets already identified antigen repetitively.
Neutrophils are granulocytes (a type of white blood cells), which contain granules. These granules have active substances which participate in inflammation and allergic reactions. Neutrophils are produced in the bone marrow. They are brought by bloodstream to the site of inflammation where they phagocytose bacteria.
Hereditary neutropenia (Trapped Neurophil Syndrome - TNS) is a hereditary autosomal recessive disease. Hereditary disease is transferred from parents to offspring. In an autosomal recessive disease a feature is transferred by a recessive allele. A monitored feature is phenotypically shown just with recessive homozygotes. Heterozygotes are with no clinical signs of the disease but they cannot be phenotypically distinguished from dominant homozygotes.
TNS is characterized by a significant lack of neutrophils in blood because they are not brought from the bone marrow into the blood circulation. This is caused by retention at the site of their origin - in the bone marrow.
It is assumed that all cases of TNS are derived from one ancestor because in the pedigrees they have the same ancestor in the past six generations.
Development of an affected dog is generally slowed down. Puppies have smaller size; and for TNS disease is typical a shape of facial skull which resembles a ferret. The individuals suffer from fever and swollen joints. Affected dogs are dying at an early age because of immune system failure.
It was found that hereditary neutropenia is very similar to Cohen syndrome, which is a human disease. Both diseases have similar symptoms; it has been proved that both are caused by mutation of the same gene.
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