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Salivary proteins of sand flies and the immune aspects of Leishmania transmission
Vlková, Michaela ; Volf, Petr (advisor) ; Schwarz, Alexandra (referee) ; Remoue, Franck (referee)
Sand flies serve as the vectors of leishmaniasis and their saliva was shown to affect the outcome of Leishmania infection by immunomodulation of the host. On the other hand, sand fly saliva contains a large scale of farmacologically active proteins that are strongly immunogenous for bitten hosts and specific anti-saliva immunity initiated by repeated sand fly feeding provides protection against Leishmania infection. Specific cell-mediated immunity was shown to be the core of the protectivity; however, our data suggests that the protective immunity has certain limitations. In mice bitten by sand flies for prolonged periods, we observed the desenzitization in term of abrogation of the protective immunity. Thus, we can speculate that the protective effect of immunity is linked solely with the short-term exposure. Nevertheless, our experiments showed that this aspect is also conditioned by the immediate infection after the protective short-term immunization. Taken together, it seems that these limitations may explain the circulation of leishmaniasis in endemic areas, even though humans and animals are frequently immunized by bites of uninfected sand flies. Repeated sand fly feeding on various hosts also promotes production of anti-saliva antibodies that reflect the intensity of exposure. We...
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Salivary proteins of sand flies and the immune aspects of Leishmania transmission
Kindlová, Michaela
Sand flies serve as the vectors of leishmaniasis and their saliva was shown to affect the outcome of Leishmania infection by immunomodulation of the host. On the other hand, sand fly saliva contains a large scale of farmacologically active proteins that are strongly immunogenous for bitten hosts and specific anti-saliva immunity initiated by repeated sand fly feeding provides protection against Leishmania infection. Specific cell-mediated immunity was shown to be the core of the protectivity; however, our data suggests that the protective immunity has certain limitations. In mice bitten by sand flies for prolonged periods, we observed the desenzitization in term of abrogation of the protective immunity. Thus, we can speculate that the protective effect of immunity is linked solely with the short-term exposure. Nevertheless, our experiments showed that this aspect is also conditioned by the immediate infection after the protective short-term immunization. Taken together, it seems that these limitations may explain the circulation of leishmaniasis in endemic areas, even though humans and animals are frequently immunized by bites of uninfected sand flies. Repeated sand fly feeding on various hosts also promotes production of anti-saliva antibodies that reflect the intensity of exposure. We...
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Methods for Leishmania parasite detection and quantificatio as a tool for study of the pathogen-vector-host interactions
Kobets, Tetyana ; Lipoldová, Marie (advisor) ; Kodym, Petr (referee) ; Kopecký, Jan (referee)
Methods for Leishmania parasite detection and quantification as a tool for study of the pathogen-vector-host interactions Leishmaniasis in human is caused by total 21 species of the intracellular protozoan parasite Leishmania, which are transmitted by about 30 species of phlebotomine sand flies. Besides human, Leishmania can infect a number of vertebrate hosts. The major host cell is the macrophage, in which parasites multiply, eventually rupturing the cell and spreading to uninfected cells. Infected monocytes and macrophages circulating in the peripheral blood are thought to be carriers of the parasite to distal sites. Depending on the infected sites of the body, there are three forms of leishmaniasis: cutaneous, mucocutaneous and visceral. Leishmaniasis is a disease for which we still lack effective, affordable and easy to use drugs. In addition, surveillance and control are also neglected. This thesis summarizes the results of several projects using different approaches for parasite load measurement in the mouse model of leishmaniasis, including two methods that were developed and optimized in our laboratory. Detection and quantification of pathogens belongs to the major topics of the research of various infectious diseases. This parameter is necessary for confirmation of the diagnosis, characterization...
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Bloodmeal digestion of phlebotomine sand flies and its effect on Leishmania development
Pružinová, Kateřina ; Volf, Petr (advisor) ; Šobotník, Jan (referee)
Leishmania development in their vectors is closely connected with bloodmeal digestion. This thesis focuses on factors affecting bloodmeal digestion, egg development and Leishmania infection within the sand fly gut. First, we compared the effect of mammalian (rabbit) and avian (chicken) blood on digestion and eggs development in Phlebotomus duboscqi. Sand flies fed on chickens had twice lower protein concentrations in the midgut and significantly lower trypsin activity compared to those fed on rabbits. The highest differences in the trypsin activity were observed during first 24 hours post bloodmeal. In addition, females fed on chickens had slower eggs development and their eggs were 10 % smaller compared to those fed on rabbits. In the second part of the thesis we tested the effect of mosquito hormone TMOF on the trypsin activity and eggs development of Lutzomyia longipalpis. Rabbit blood with TMOF (28 mg/ml) was presented to the females via a membrane feeding system. Sand flies fed on blood with TMOF had 15 - 35 % less trypsin activity than control females fed on only rabbit blood. In addition, females fed on blood with TMOF had developed 30 % less eggs and their eggs were 12 - 24 % smaller compared to control group. However the effect of TMOF we observed was lower than that described previously...
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The development of Leishmania in the insect vector of the genus Phlebotomus
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Bilej, Martin (referee)
The work was focused on the study of various aspects of development of Leishmania in vectors of genus Phlebotomus and can be divided into two main parts. In the first part, we studied the competition of Leishmania major and L. turanica during their development in the sand flies who are natural vectors of L. major using the experimental co-infections of fluorescently marked promastigots of these two species. While both leishmania species developed similar in the intestine of Phlebotomus papatasi, L. turanica prevailed in P. duboscqi in the late stages of infection. The fluorescent marking of Leishmania should allow us also to study possible genetic exchange between species at different stages of Leishmania infection (2nd, 9th and 14th day after the infective feeding). Using the flow cytometry (FACS) we have repeatedly identified dozens of objects emitting red and green signals in the intestinal homogenates of co-infected sand flies, however further analysis with confocal microscope disproved these objects as the hybrid promastigots of Leishmania. In the second part of this thesis we investigated the role of L. major genes HASP and SHERP, which lies on the locus LmcDNA16 and are expressed exclusively in metacyclics. We used mutant lines KO (lacking the locus LmcDNA16) and HASPB (KO line with gene...
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Macrophages in leishmania - sand fly - host interaction
Kratochvílová, Tereza ; Kolářová, Iva (advisor) ; Fialová, Anna (referee)
Sand flies (order Diptera) are vectors of Leishmania parasites (Trypanosomatida), which are inoculated into the host skin together with the vector saliva. Sand fly saliva plays the important role in the Leishmania transmission; in naive host it supresses the host immune response assisting Leishmania to establish the infection, while in repeatedly bitten host it elicits a protective immune response. The submitted thesis focuses on the effect of sand fly saliva on macrophages, the key cells in the infection control. In the first part of the thesis we established a laboratory model L. major - P. papatasi - Balb/c to describe the protective effect of saliva immunization on Leishmania infection development. Immunized mice were protected against Leishmania infection which was reflected in the ear lesion size, parasite load in the ear dermis and draining lymph nodes but also in cytokine production. On the contrary, produced lower amount of nitric oxide, while arginase activity was comparable with nonimmunized group. The IgG antibodies against saliva served as a marker of exposure to sandflies while IgG antibodies against Leishmania antigens served as a marker of infection severity. The experiments were aimed on the possibility of cross-protectivity in Balb/c mice against L. major between closely related...
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Sex in Trypanosomatids
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Čepička, Ivan (referee)
3 Abstrakt Rody Leishmania a Trypanosoma jsou původci vážných lidských onemocnění: leishmaniózy a trypanosomózy. Dlouhá léta nebyly u těchto parazitů nalezeny přesvědčivé důkazy o genetické výměně, a proto byly rody Trypanosoma a Leishmania považovány za klonálně se rozmnožující, a to binárním štěpením jako většina prvoků. Výzkum ztěžovaly i skutečnosti, že pohlavní dimorfismus není patrný a chromosomy nekondenzují, tudíž nejsou viditelné. Nicméně klonální model začaly zpochybňovat pozorování přirozeně se vyskytujících hybridních druhů. Nejdříve byla existence sexu popsána u trypanosom a to prvním přímým důkazem hybridů T. brucei, získaných po společném přenosu rodičů mouchou tsetse. U leishmanii byl důkaz poskytnut na základě dvojitě rezistentních hybridů a sexuální výměna podstupovala stejný meiotický proces jako T. brucei. Byli pozorovaní přirozeně se vyskytující hybridi Nového i Starého světa jak u rodu Viannia, tak i u rodu Leishmania. Otázkou dalších výzkumů bylo, jaký je mechanismus genetické výměny, ale odpověď dodnes není jasná. Klíčová slova: genetická výměna, Trypanosoma, Leishmania, klonalita, meióza, GFP, přenašeč Abstract Genera Leishmania and Trypanosoma are agents of serious human diseases: leishmaniasis and trypanosomózy. For many years these parasites were considered clone-replicating by...
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Phlebotomine saliva: the perspecives of application of some of their recombinantly produced proteins
Rožníčková, Lucie ; Zídková, Lenka (advisor) ; Štáfková, Jitka (referee)
Sandflies (Diptera: Phlebotominae) are the vectors of one of the most significant tropical diseases, leishmaniosis. Their saliva contains substances with antihemostatic, vasodilatory and immunomodulatory effect. These substances are of great significance in transmission of leishmaniosis, because of their ability to influence the host immune response. Naive host saliva modulate the immune system to such an extent, that it reduces an infective dose and increases the pathogenicity of leishmaniosis. On the other hand, a repeatedly exposed host generates a cellular and antibody immune response, which is able to make the initial development of infection impossible. Repeated exposure of individual sandflies lead to formation of antibodies against components of saliva, which may play a role as a rate exposure indicator and thus the risk of leashmaniosis infection. These proteins and glycoproteins can be produced by recombinant DNA technology. Recombinant vaccine could help in the fight against leishmaniasis. Keywords: saliva, sand fly, leishmania, recombinant proteins.
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Macrophages and nitric oxide in leishmania - sandfly - host interactions
Kratochvílová, Tereza ; Fialová, Anna (referee) ; Kolářová, Iva (advisor)
Leishmania reside fagolysosome of macrophages immediately after their entry to host where they multiply and consequently infect other macrophages or eventually other cells. A synthesis of a reactive reactant of oxygen and nitrogen is one of the mechanisms that some mammal cells are equipped with and that also contributes to eradication of leishmania. Nitric oxide rising during a metabolic change of L-arginine under the catalysis of NO synthase is of a large importance. Beyond cytotoxic function, nitric oxide is involved in signalling pathways for a neurotransmission (nNOS) and vasorelaxation (eNOS). Not all types of macrophages have ability to produce NO (iNOS). It is a heterogeneous group differing in immunological function and also in physiology. A group of classical activated macrophages represents an effective APC capable of efficient killing of intracellular pathogens. In addition to NO, they also secrete an inflammatory cytokines, which evolve an immune reaction towards to Th1. Contrary to this, a group of alternative activated macrophages is not capable of any efficient antigen presentation and nitric oxide production but produces L-ornithine, which is a precursor of polyamines, which leishmania utilizes for its own intracellular growth. For the mouse model, status of resistance and/or...
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