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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Preparation of expression vectors for NKp65 and KACL, new members of human NK cell receptor family
Mikulová, Barbora ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells create an important part of innate immune system. Their importance lies in their ability to recognize and kill abnormal cells, especially tumour cells and virally infected ones, without previous activation. To recognize their targets, NK cells use a wide variety of surface receptors, both activating and inhibitory. If a ligand binds to an NK receptor, immune response is triggered. Examples of such ligand-receptor pairs are NKp80-AICL and NKRP1-LLT1 on human lymphocytes. Another ligand-receptor system of this kind is NKp65 and KACL, two recently discovered lectin receptors on human immunocytes. KACL is the last and most recently characterized member of CLEC2 receptor family in humans. Its expression is almost exclusively restricted to skin. NKp65, a close relative of NKp80, is a glycoprotein which stimulates NK92MI cell cytotoxicity upon KACL engagement. NKp65 has been shown to bind to KACL with a fairly high affinity by surface plasmon resonance measurement. This thesis aims at describing the cloning of expression vectors coding for NK cell receptors NKp65 and KACL, expression of these proteins in HEK293T cell line and their purification. Keywords: NKp65, KACL, NK cell, lectin, receptor, plasmid (in Czech)
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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Chicken antibodies as a tool of passive immunization against microbial diseases of respiratory tract
Růžička, Martin ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Pseudomonas aeruginosa and Burkholderia cepacia are opportunistic human pathogens. Hazards pose to immunocompromited patient groups, most of whom are patients suffering from cystic fibrosis, for which is the infection caused by Pseudomonas aeruginosa or Burkholderia cepacia, often lethal. Bacteria exhibit resistance to most antibiotics and the immunization of patients, paradoxically, worsens the patients' condition. Hen antibodies, unlike mammalian do not activate the complement cascade and do not cause the inflammatory response, therefore, seem to be a suitable tool to protect high risk populations as a means of passive immunization. In this work were prepared specific chicken antibodies against significant virulent factor - bacterial lectins PAIIL and BClA responsible for adhesion. Antibody specificity was demonstrated by ELISA and Western blot. Antibodies were affinity purified and cleaved to fragments. Pneumocytes type II from the lungs of patients with Cystic Fibrosis were isolated and cultured. Tests of the adhesion of bacteria cells were performed on them using ELISA. As an alternative model rat pneumocytes and tumor cell line A549 has been used. Prepared antibodies specifically detect bacterial lectins on the surface of bacteria. Antibody has been shown to reduce adhesion of bacteria to...
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PCR based assay for detection garden pea lec gene
Vráblík, Aleš ; Hodek, Jan ; Ovesná, Jaroslava
The method was developed and verified by staff of National reference laboratory for GMO identification and DNA fingerprinting suited for governmental control laboratories and private laboratories that analyze food and feed derived from various plant matrices. Method can be applied for quantification of GM pea in a sample, when reference gene is used as a standard. The method describe internal garden pea specific gene, lektin in this case, detection using PCR and real-time PCR. The general principle of the assay relies on lektin specific sequence presence that represents a species specific gene of garden pea. In many matrices DNA is damaged so amplification of short stretches of DNA is used. After amplification of target exploiting newly developer species specific gene primers PCR products are separated by electrophoresis in agarose gel. Resulting band is visualized by UV light and its position is compare with size standard. Alternatively, real-time PCR and ABI platform in combination with SYBR® Green can be used. Reaction parameters are described and specificity of reaction was verified. LOD as well as LOQ was defined.
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Identification and biochemical characterization of lectins in the hemolymph of three species of tick in the genus \kur{Rhipicephalus}
FIŠER, Miroslav
Lectins are tissue specific carbohydrate binding proteins with possible functions in invertebrate immunity and pathogen transmission. The main goal of this study was to identify hemolymph lectins in three different tick species. Three proteins with molecular weights of 58 kDa, 75 kDa and 180 kDa were detected in all investigated species using antibodies directed against hemagglutination activity of Rhipicephalus appendiculatus hemolymph. These proteins were characterized by biochemical methods such as Schiff/periodate staining, lectin blotting, enzymatic deglycosylation, hemagglutination analysis, immunoblotting, and mass spectrometry.
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Isolation and biochemical characterisation of FREP proteins from \kur{Dermacentor marginatus}.
ŠTĚRBOVÁ, Jarmila
Fibrinogen-related proteins (FREP) are lectins with possible functions in invertebrate immunity. Previously, a FREP protein from the hemolymph of the tick Ornithodoros moubata, Dorin M, was isolated and characterised in our laboratory. In this study, four FREP proteins named DMFREP1, DMFREP2, DMFREP3, and DMFREP4 were detected with anti-Dorin M antibodies in the hemolymph of the tick Dermacentor marginatus. These FREP proteins were characterised by biochemical methods, purified, antibodies directed against these proteins were produced and the proteins were localised to tick issues by immunofluorescence.
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