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Diversity of methods used for characterization of molluscan hemocytes
Jindrová, Zuzana ; Horák, Petr (advisor) ; Skála, Vladimír (referee)
Hemocytes are the main immune cells of invertebrates; therefore they can be found in molluscs, too. They differ both in morphology and function. The two generally accep- ted morphological types, granulocytes and hyalinocytes, vary in the level of phagocy- tosis and encapsulation, production of reactive oxygen species and nitrogen oxide, and presence of some enzymes. There is an array of methods by means of which hemocy- tes can be characterized. Microscopy serves particularly for study of morphology. An- tigens localized on the surface can be determined by monoclonal antibodies or lectin probes. Hemocytes can be divided on the basis of cell size and granularity using gra- dient centrifugation or flow cytometry. Production of nitrogen oxide and reactive oxy- gen species is monitored by adding appropriate substrate which changes its proper- ties after reaction with the radical. It may become fluorescent, change absorbance of the solution or form a visible precipitate. Another possibility is the use of chemilu- miniscence. The objective of hemocyte research is to explain mollusc-pathogen inter- action. 1
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Effect of ageing on haematological and immunological traits in great tit (Parus major)
Krajzingrová, Tereza ; Vinkler, Michal (advisor) ; Remeš, Vladimír (referee)
v III. Abstract (EN) According to the Disposable soma theory, senescence is mainly influenced by adaptive allocation of resources among the physical growth, reproduction and self-maintaining processes, which depend on expected survival of an individual. The amount of allocated resources may be influenced by external environmental factors such as heavy metal pollution or by inner factor e.g. reproduction. The aim of my diploma thesis was to clarify the relationships between senescence, selected haematological traits, reproduction and efficiency of innate immunity mechanism. Moreover, we are interested in the role of heavy metal pollution in ageing and haematology. The heterophiles to lymphocytes ratio (H/L) was used as a marker of long-term stress caused by these physiological expenses. This study summarizes partial data collected within the long-term research of great tits (Parus major) in Prague, Czech Republic, I participated in. We have more than 300 samples of repeatedly captured individuals at our disposal. The structure of this unique dataset allows us to observe changes in the traits of our interest during an avian lifespan. Our results show that during aging, males experience a noticeable increase in the H/L ratio compared to females. Although we have not found the effect of age on the brood size,...
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Diversity of methods used for characterization of molluscan hemocytes
Jindrová, Zuzana ; Skála, Vladimír (referee) ; Horák, Petr (advisor)
Hemocytes are the main immune cells of invertebrates; therefore they can be found in molluscs, too. They differ both in morphology and function. The two generally accep- ted morphological types, granulocytes and hyalinocytes, vary in the level of phagocy- tosis and encapsulation, production of reactive oxygen species and nitrogen oxide, and presence of some enzymes. There is an array of methods by means of which hemocy- tes can be characterized. Microscopy serves particularly for study of morphology. An- tigens localized on the surface can be determined by monoclonal antibodies or lectin probes. Hemocytes can be divided on the basis of cell size and granularity using gra- dient centrifugation or flow cytometry. Production of nitrogen oxide and reactive oxy- gen species is monitored by adding appropriate substrate which changes its proper- ties after reaction with the radical. It may become fluorescent, change absorbance of the solution or form a visible precipitate. Another possibility is the use of chemilu- miniscence. The objective of hemocyte research is to explain mollusc-pathogen inter- action. 1
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Laboratory diagnosis of disorders of phagocytosis
POLÍVKOVÁ, Ivona
Phagocytosis is one of the oldest processes of absorbing particles like amoeba, which is one of the basic mechanisms of the immune system to defend the body against infections. Failure of these processes is clinically manifested like immune deficiency and it can cause a very serious complication which could leads to the death of the patient. Phagocytosis is performed by specialized cells (macrophages, dendritic cells, neutrophils and other phagocytic myeloid cells) which are able to absorb the target particles, especially microorganisms, dead cells and foreign objects. These processes are essential for stability of homeostasis. Absorption of micro-organisms leads to activation of adaptive immunity response, elimination of apoptotic and destroyed cells and starts the reparation processes of damaged tissue. Phagocytosis as a complex process can be divided into several phases: active movement of phagocytes to the inflammation zone, adherence, ingestion and intracellular degradation which leads to the killing of pathogens. There are two mechanisms of killing pathogens. First is independent on oxygen where antimicrobial substances are stored in azurophilic granules which could be released into phagolysosom. Second mechanism is oxygen-dependent called oxidation (respiratory) flare which leads to the formation of biologically active mediators where oxygen molecules have considerable oxidation potential. Defects in phagocytic system are mainly caused by low number or malfunction of neutrophils which leads to severe infections mainly caused by staphylococci, Enterobacteriaceae or fungi. Among malfunctioning of phagocytosis processes belongs LAD I and LAD II syndrome. The other possible disorder of phagocytic malfunction is defect in enzymes. The NADPH oxidase is necessary for bacterial lyses mechanism of phagocytes and lack of these enzyme caused serious inherit disorder called chronic granulomatous disease. Lack or completely missing of enzyme myeloperoxidase stored in the azurophilic granules of neutrophils and monocytes is more and more common autosomal recessive disorder of phagocytosis. In this diploma work I focused on detection of these rare and serious disorders defects using flow cytometry to detect respiratory burst activity of neutrophils in blood samples. This laboratory procedure is called a burst test. In this test we quantitatively evaluate the respiratory burst activity of granulocytes in heparinized blood samples using the flow cytometery. Principle of this method is an oxidation-reduction reaction of dihydrorhodamine 123 to green fluorescent rhodamine 123 using peroxide, hydroxyl radicals and superoxide anions which are activated by respiratory burst. This process is one of the significant characteristic of phagocytic cells which are characterized by multi-stage activation of NADPH oxidase. This oxidase catalyzes electron reduction of molecular oxygen to superoxide. This step plays a very important role in our immune system and allows to kill and degrade particles and bacteria in phagocyte cells. Since 2009 - 2012 I analyzed the results of 611 patients who were tested for respiratory burst of neutrophils on a request of their physician. Among all of these results we obtain only two positive results of reduction of stimulation index (SI) and significant decrease percentage of activated granulocytes. Both of these parameters: reduced percentage of (activated granulocytes stimulated by PMA and E. coli) and low stimulation index pointing to a potentially serious disorder of phagocytosis mechanism. In specific tests of these cases proved the enzyme defect in phagocytosis mechanism which is the most common disorder in this type of immunodeficiency. These results indicate that the major primary defects of phagocytosis are very rare and their detection is usually in childhood for suspicion of primary immunodeficiency. This test is very helpful for discovering a prime immunodeficiency.
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