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Background and objectives: The relationship between air pollen quantity and the sensitization of allergic patients is crucial for both the diagnosis and treatment of allergic diseases. Weather conditions influence the distribution of allergenic pollen and increases in pollen concentration may negatively affect the health of allergic patients. The aim of this study was to analyze the implementation of allergen immunotherapy with regard to air pollen concentration. Material and Methods: Here we examined the relationship between Betula air pollen concentration and the usage of Betula verrucosa allergen immunotherapy in Serbia. Examination covered the period from 2015 to 2018. Measurement of airborne pollen concentration was performed with Lanzoni volumetric pollen traps. The evidence of the usage of sublingual allergen immunotherapy (SLIT) was gathered from patients with documented sensitization to specific pollen. Results: During this period tree pollens were represented with 58% ± 21% of all measured air pollen species, while Betula pollen represented 15% ± 8% of all tree pollens. Betula pollination peaked in April. Allergen immunotherapy to Betula verrucosa in Serbia is entirely conducted as sublingual immunotherapy and represents 47.1% ± 1.4% of issued tree pollen SLIT. The use of pollen SLIT increased by 68% from 2015 to 2018, with an even greater increase in usage recorded for Betula SLIT—80%. Conclusions: This analysis shows a clear causative relationship between pollination and the type/prevalence of applied allergen immunotherapy. Information about the flowering seasons of allergenic plants is very important for people who suffer from allergy, for clinical allergologists, as well as for governing authorities. The presented data is of practical importance to the proper timing of immunotherapy initiation and of importance for urban landscaping. The obtained data can be the starting point for the instatement of a thorough epidemiological study and the inclusion o

A disposable acetaminophen biosensor based on inkjet-printed CNT electrodes (IJPCNT) modified with amidase/cerium dioxide@graphene nanoribbons composite was developed (ACeO2@GNR/IJPCNT). The enzyme amidase A was used for the first time as a recognition element. Inkjet-printed CNT electrodes served as a basis for the construction of a biosensor that enables droplet detection using 5 μL sample volume. The biosensor showed high selectivity, sensitivity, a low detection limit of 0.18 μM and a wide working linear range from 1 to 100 μM. The proposed approach allows fast and reliable detection of acetaminophen in biological fluids with negligible matrix effect and remarkable reproducibility. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

A disposable acetaminophen biosensor based on inkjet-printed CNT electrodes (IJPCNT) modified with amidase/cerium dioxide@graphene nanoribbons composite was developed (ACeO2@GNR/IJPCNT). The enzyme amidase A was used for the first time as a recognition element. Inkjet-printed CNT electrodes served as a basis for the construction of a biosensor that enables droplet detection using 5 μL sample volume. The biosensor showed high selectivity, sensitivity, a low detection limit of 0.18 μM and a wide working linear range from 1 to 100 μM. The proposed approach allows fast and reliable detection of acetaminophen in biological fluids with negligible matrix effect and remarkable reproducibility.

Celobioza – dehidrogenaza poreklom iz Phanerochaete chrysosporium, gljive bele truleži, pripada ekstracelularnim oksidoredukcionim enzimima i katalizuje oksidaciju β – 1,4 – glikozidno vezanih oligosaharida poput celobioze i laktoze. Oksidacijom laktoze dolazi do formiranja laktobionske kiseline koja pronalazi veliku primenu u farmaceutskoj i kozmetičkoj industriji gde se koristi prilikom distribucije lekova i za hidrataciju kože kao sastavni deo različitih krema, gde zajedno sa hijaluronskom kiselinu ima ulogu u smanjenju bora.Celobioza – dehidrogenaza prilikom oksidacije laktoze ili celobioze, kao prirodnih supstrata, katalizuje redukciju jednog dvoelektronskog ili dva jednoelektronska akceptora elektrona. Jedan od najkorišćenijih dvoelektronskih akceptora elektrona je upravo dihlor fenol indofenol (DCIP), dok jednoelektronski akceptori elektrona mogu biti citohrom c, ABTS, ali i Fe3+ i Mn3+ joni. Redukcijom Fe3+ jona u prisustvu molekulskog kiseonika dolazi do formiranja vodonik peroksida i posredstvom Fentonove reakcije do generisanja hidroksil radikala.Polazeći od ove činjenice, iskoristili smo upravo Fentonovu reakciju za razvoj fluorescentnog eseja za visoko efikasnu pretragu biblioteka gena celobioza – dehidrogenaze, baziranog na detekciji proizvedenih hidroksil radikala fluorogenom probom aminofenil – fluoresceinom (APF).Primena celobioza – dehidrogenaze u konstruisanju biosenzora i biogorivnih ćelija leži upravo u njenoj sposobnosti da katalizuje oksidaciju laktoze, celobioze i β – 1,4 – vezanih oligosaharida do odgovarajućih laktona koji potom spontano hidrolizuju do aldonskih kiselina. Enzimi koji nalaze primenu u konstruisanju biosenzora i biogorivnih ćelija, moraju da zadovoljavaju nekoliko kriterijuma,odnosno moraju da imaju veliku osetljivost i supstratnu specifičnost, ali i da pokazuju povećanu stabilnost..., Cellobiose dehydrogenase from Phanerochaete chrysosporium, a white rot fungus, belongs to the extracellular oxidoreductive group of enzymes and catalyzes the oxidation of the β – 1,4 – glycoside bond of oligosaccharides such as cellobiose and lactose. During oxidation of lactose, formation of lactobionic acid occurs which has many applications in pharmaceutical and cosmetic industry. Such applications include the distribution of medicine, a component responsible for skin hydration and when combined with hyaluronic acid as an agent against wrinkles.During oxidation of lactose or cellobiose by cellobiose dehydrogenase, reduction catalysis occurs of one two electron or two one electron acceptors. One of the most utilized two electron acceptors is DCIP, while one electron acceptors are usually cytochrome c, ABTS, Fe3+ and Mn3+ ions. During reduction of Fe3+ ions in the presence of molecular oxygen, H2O2 is formed and due to the Fenton reaction formation of hydroxyl radicals occurs. Due to this occurrence we wanted to use the Fenton reaction in order to develop a fluorescent assay based on the production of hydroxyl radicals and the fluorescence of aminophenyl fluorescein (APF). This would allow us to efficiently analyze cellobiose dehydrogenase gene libraries.With this fact in mind, the Fenton reaction was used to develop a fluorescent assay for the high throughput screening of cellobiose dehydrogenase genes, based on the detection of hydroxyl radicals with the fluorescent probe APF.The possible application of cellobiose dehydrogenase in the construction of various biosensors and biofuel cells is due the its ability to catalyze the oxidation of lactose, cellobiose and similar β – 1,4 – oligosaccharides do their corresponding lactones which then spontaneously hydrolyze to aldonic acids. Enzymes used in suchapplications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity...

U ovoj doktorskoj disertaciji je ispitan potencijal HAP za imobilizaciju industrijski značajnih enzima na primeru 4 lipaze i 4 glikozidaze različitog porekla, strukture i supstratne specifičnosti. Od testiranih enzima za HAP se najefikasnije vezuju lipaza iz Candida rugosa (CRL) i β-galaktozidaza (laktaza) iz Aspergillus oryzae (AOL). Oba enzima se imobilizuju na HAP jednostavnom, brzom i efikasnom fizičkom adsorpcijom po mehanizmu metal-koordinatne veze. Kombinovanjem bioinformatičkog pristupa i molekulskog modelovanja za oba enzima su identifikovani i opisani površinski HAP-vezujući motivi.Imobilizacija na HAP je uzrokovala promenu supstratne specifičnosti CRL ka supstratima sa kraćim alkil-nizom i drastično povećanje stabilnosti i aktivnosti enzima u metanolu. Ove karakteristike imobilizata CRL na HAP (CRL-HAP) su prvi put u literaturi primenjene za sintezu metil-acetata, estra sa mirisom jabuke, koga slobodna CRL sintetiše u zanemarljivom prinosu. Pažljivim odabirom rastvarača i supstrata je, takođe po prvi put pokazano kako ovaj popularni i jeftin enzim, u slobodnom ili imobilizovanom obliku, može da se primeni i za efikasnu sintezu kapsinoida direktnom esterifikacijom vanilil-alkohola (VA) i masnih kiselina (MK) i transesterifikacijom kokosovog ulja.Efikasnost imobilizovanog preparata AOL (AOL-HAP) je ispitana u sintezi bioaktivnih supstanci, vanilil-galaktozida (VG) i galakto-oligosaharida (GOS). Po efikasnosti u sintezi ovih proizvoda preparat AOL-HAP se našao u rangu sa kovalentno imobilizovanim preparatima AOL. Jedna ista šarža AOL-HAP može se iskoristiti u 10 uzastopnih reakcionih ciklusa sinteze VG, odnosno GOS, sa poluživotom od 15 h., Potential of hydroxyapatite (HAP) as immobilization carrier for industrially important enzymes was examined in a study, comprising of 4 lipases and 4 glycosidases of different origin, structure and substrate specificity. Lipase from Candida rugosa (CRL) and β-galactosidase (lactase) from Aspergillus oryzae (AOL) exhibited the most efficient binding to HAP. Both of the enzymes were immobilized on HAP by simple, fast and efficient physical adsorption through formation of metal-coordinative bond. Furthermore, by the combining of bioinformatic approach and molecular modeling, HAP-speciffic superficial motif on both selected enzymes was identified and described for the first time in the literature.Immobilization on HAP has shifted substrate specificity of CRL towards shorter alkyl-chains, coupled with significant increase in stability and activity of CRL in methanol. These characteristics of immobilized Candida rugosa lipase preparation (CRL-HAP) were sucessfully implemented in synthesis of methyl-acetate, an apple flavour for the first time in the literature. Free CRL synthesized methyl-acetate in very low yield, because of methanol-induced inactivation. By a meticulous selection of organic solvent and susbtrates, it was shown also for the first time that this popular and cost-effective microbial lipase, free or immobilized on HAP, can be used for efficient synthesis of capsinoids in two distinctive reactions: direct esterification of vanillyl-alcohol (VA) with free fatty acids and transesterification of coconut oil.Efficiency of AOL immobilized on HAP (AOL-HAP), was evaluated in synthesis of bioactive compounds: vanillyl-galactoside (VG) and galacto-oligosaccharide (GOS) probiotics. Efficiency of AOL-HAP in synthesis of these compounds was in a good correlation with the procedures in which covalently immobilized AOL was employed. Immobilized AOL preparation can be used in maximum 10 consecutive reaction cycles during VG and GOS synthesis, with the half-life of 15 hours

Impairment of the intestinal barrier is one of the key events in the initiation of the sensitization process in food allergy. The aim of this study was to explore the effects of kiwifruit allergen Act d 1 on intestinal permeability and tight junction protein (TJP) gene expression in vivo and to explore its potential to activate the NF-ĸB signaling pathway and to regulate expression of epithelial pro-allergenic cytokines. Influences of Act d 1 on TJP gene expression and pro-allergenic cytokines in the mouse intestine was analyzed by qPCR upon allergen administration by oral gavage. The effect on the in vivo intestinal permeability was assessed in ELISA by measuring the translocation of β-lactoglobulin (BLG) into circulation. The capacity of Act d 1 to activate the NF-ĸB pathway was tested in HEK293 cells by fluorescent microscopy and flow cytometry. Administration of Actinidin (Act d 1) increased intestinal permeability to the BLG. This was accompanied by changes in gene expression of TJP mRNA and pro-allergenic cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) compared to the control. Act d 1 reduced TEER of the HEK293 monolayer, was positive in an NF-ĸB-reporter HEK293 cell assay, and induced secretion of TSLP. These findings shed more light on the molecular events in the sensitization process of kiwifruit but possibly also of other protease food allergens.

Gastrointestinalna barijera izgrađena je od veoma kompleksne mreže ćelija, mukusa i drugih važnih komponenti imunog sistema. Epitelni sloj ćelija gastrointestinalnog trakta predstavlja važnu komponentu urođene imunosti koja na dinamičan način održava homeostazu. Međutim, narušavanje integriteta gastrointestinalne barijere može se nepovoljno odraziti na organizam. Takav poremećaj dovodi do razvoja alergije na hranu, koja predstavlja neprimeren odgovor ćelija imunog sistema na proteine hrane. Epitelne ćelije gastrointestinalnog trakta, kao posledica kontakta sa alergenom hrane, utiču na komponente imunog sistema (smeštene u sloju lamina propria) u pravcu razvoja Th2 ćelijskog odgovora i proizvodnje specifičnih IgE antitela.Alergije na hranu nastaju kao posledica narušavanja imunološkog odgovora označenog kao oralna toleranca na proteine hrane koji podrazumeva kako proizvodnju specifičnih regulatornih ćelija tako i sintezu određenih signalnih molekula. Alergijske reakcije izazivaju širok spektar kliničkih simptoma i mogu se manifestovati od vrlo blagih reakcija do fatalnih, kao što je anafilaktički šok.Alergeni hrane koji poseduju proteaznu aktivnost u gastrointestinalnom traktu mogu da dovedu do narušavanja integriteta intestinalne barijere i time izazovu neprimerenu reakciju imunog sistema. Osim što pokazuju otpornost na proteolizu i drastične uslove digestivnog trakta, proteaze iz hrane mogu da zadrže i imunogena svojstva. Alergija na kivi je veoma rasprostranjena i značajan je uzročnik alergija na voće. Glavni alergen ploda zelenog kivija, aktinidin (Act d 1), je cistein proteaza (EC 3.4.22.14) koja je ujedno najzastupljeniji i najvažniji alergen kivija, čineći preko 50 % ukupnih solubilnih proteina kivija.U ovoj disertaciji ispitivan je efekat aktinidina i posledice njegove proteazne aktivnosti na epitelne ćelije gastrointestinalnog trakta..., The gastrointestinal (GI) barrier consists of a complex network of cells, mucus and other important components of the immune system. The epithelial cell layer of the gastrointestinal tract (GIT) is an important component of innate immunity, which dynamically maintains homeostasis. Therefore, disruption of the integrity of the gastrointestinal barrier can adversely affect the body. Food allergy, an inappropriate response of immune cells to food proteins, is an example of homeostasis disruption. After contact with a food allergen, gastrointestinal epithelial cells stimulate immune cells of lamina propria to induce Th2 cell response and produce specific immunoglobulin E (IgE). Oral tolerance is a normal response of the immune system to food proteins, which includes the production of specific regulatory cells and signal molecules. Disruption of oral tolerance gives rise to food allergy with a wide array of clinical symptoms from mild to life-threatening anaphylaxis. Food allergens with protease activity can impair the integrity of the gastrointestinal barrier and thus cause an inappropriate response of the immune system. These allergens are resistant to proteolysis in the harsh environment of the digestive tract and can also be immunogenic. Allergy to kiwi is among the most common fruit allergies with worldwide prevalence. Actinidin (Act d 1), cysteine protease (EC 3.4.22.14) is the most abundant allergen of green kiwi-fruit. In this dissertation, the effect of Act d 1 proteolytic activity on gastrointestinal epithelial cells was investigated in vitro and in vivo. It is shown that the proteolytic activity of actinidin impairs the integrity of the gastrointestinal barrier in C57BL/6 mice. The same was confirmed in human intestinal epithelial Caco-2 cells. Actinidin hydrolyzes tight junction (TJ) proteins in situ (occludin, ZO 1, claudin 3 and E-cadherin) in Caco-2 cells and 2D organoids derived from murine intestinal epithelial cells. Additionally, active actinidin affec

Etiologija tiroidnih oboljenja je nedovoljno prouĉena i najnoviji literaturni podaci ukazuju da bi metali mogli da igraju kljuĉnu ulogu u patogenezi velikog broja poremećaja, ukljuĉujući i bolesti tiroidne ţlezde. S tim u vezi, cilj ove studije bio je da se odredi sadrţaj esencijalnih i toksiĉnih metala u zdravim tiroidnim tkivima (ZTT) i napravi komparativna analiza sa sadrţajem metala u tiroidnim tkivima sa dijagnostifikovanim benignim i malignim oboljenjima. Dodatni cilj studije bio je sagledavanje metalomike u zavisnosti od bioloških i kliniĉko-patoloških parametara, kao i pronalaţenje najznaĉajnijeg metala (ili njegovog odnosa sa drugim metalom) koji bi mogao da razdvoji ispitivane grupe i obezbedi informacije o molekulskoj osnovi patofizioloških promena ispitivane bolesti tiroidne ţlezde na tkivnom nivou. Uzorci tkiva za analizu metala sakupljeni su nakon operacije tiroidne ţlezde. U istraţivanju je uĉestvovalo 200 pacijenata, od toga 21 pacijent sa Hašimotovim tiroiditisom (HT), 62 pacijenta sa koloidnom strumom (KS), 36 pacijenata sa benignim tumorima (BT) i 81 pacijent sa malignim tumorima (70 sa papilarnim karcinomom, 8 sa folikularnim i troje sa medularnim karcinomom). Od svakog pacijenta sa dijagnostifikovanim tumorom uzorkovano je i ZTT sa najveće moguće udaljenosti od primarnog tumora, koji je bio jasno demarkiran (n = 117). U svakoj patologiji ispitan je sadrţaj metala u zavisnosti od pola, uzrasta, konzumiranja duvana i veliĉine tiroidnog nodusa. Metalomika papilarnog karcinoma tiroidne ţlezde dodatno je ispitana u zavisnosti od patohistološkog (PH) tipa tumora, prethodne istorije tiroidnog oboljenja u porodici, „TNM“ klasifikacije, prisustva invazije tiroidne kapsule, intratiroidne multicentriĉne diseminacije i retrosternalnog tiroidnog rasta. Uzorci tkiva razoreni su mikrotalasnom digestijom i koncentracija elemenata, mangana (Mn), nikla (Ni), bakra (Cu), cinka (Zn), arsena (As), selena (Se), kadmijuma (Cd), olova (Pb), torijuma (Th) i uranijuma (U, The etiology of thyroid diseases is not very well clarified and the latest literature data indicated that metals could play a key role in the pathogenesis of a great number of disorders, including the thyroid gland diseases. In this regard, the aim of this study was to determine the content of toxic and essential metals in the healthy thyroid tissues (HTTs), as well as to compare the content of metals in tissues with diagnosed benign and malignant thyroid gland diseases. The further aim of this study was to investigate metallomics according to the biological-, clinical- and pathological-parameters, as well as to find the most significant metal (or its ratio to another metal) that could separate examined groups, as well as to provide additional information on the molecular basis of pathophysiological changes of the examined thyroid disease at the tissue’s level. Tissue samples for metal analysis were collected after thyroid surgery. The study included 200 patients, of which 21 patients with Hashimoto’s thyroiditis (HT), 62 patients with colloid goiter (CG), 36 patients with benign tumors (BTs) and 81 patients with malignant tumors (70 with papillary carcinoma, 8 with follicular and three with medullary thyroid carcinoma). HTT was sampled from each patient with diagnosed tumor from the greatest possible distance from the primary tumor, which was well demarked (n = 117). In each pathology, the content of metals was investigated according to sex, age, smoking habits and goiter size. Metallomics of the papillary thyroid carcinoma (PTC) was further examined based on the pathohistological (PH) type of tumor, previous history of any thyroid disease in the family, TNM classification, capsular invasion, intrathyroid dissemination, and the retrosternal thyroid growth. Tissue samples were decomposed by microwave digestion and the concentration of elements, manganese (Mn), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), lead (Pb), thorium (Th) and

Introduction: In the Serbian population allergy to weed pollen is the most common type of pollen-associated allergy, ahead of grass and tree pollens. Besides causing discomfort, allergy to pollen is often associated with rhinitis and asthma. Allergen-specific immunotherapy (ASIT) is the only treatment that can lead to potential long-term immune modification while reducing development of new sensitization and halting disease progression. The aim of the present study is to evaluate the efficacy of sublingual immunotherapy (SLIT) to weeds in the adult patient population using vaccine produced by the local Serbian Torlak Institute for virology, vaccines and serum. Methods: Adult patients with a clinical history of allergic rhinitis with and without asthma were included in the study. IgE-mediated sensitization to grass, tree and weed pollens was confirmed by skin prick testing and/or positive specific IgE. Patients were divided into two groups: patients with allergy to tree and grass pollen and patients with allergy to weeds. All patients received SLIT for three years, either with or without additional symptomatic therapy. Results: Three-year SLIT therapy led to significant improvement in several parameters, including skin-prick reactivity, decrease in specific IgE and use of symptomatic therapy, with mild adverse effects and high patient satisfaction concerning therapy. Conclusion: Three-year SLIT is a safe and efficient treatment option for respiratory allergy to weeds. Further observations in a larger number of patients could provide a better epidemiological evaluation of SLIT, but the positive effects we observed in our study may be considered representative despite the small number of patients.

Etiologija tiroidnih oboljenja je nedovoljno prouĉena i najnoviji literaturni podaci ukazuju da bi metali mogli da igraju kljuĉnu ulogu u patogenezi velikog broja poremećaja, ukljuĉujući i bolesti tiroidne ţlezde. S tim u vezi, cilj ove studije bio je da se odredi sadrţaj esencijalnih i toksiĉnih metala u zdravim tiroidnim tkivima (ZTT) i napravi komparativna analiza sa sadrţajem metala u tiroidnim tkivima sa dijagnostifikovanim benignim i malignim oboljenjima. Dodatni cilj studije bio je sagledavanje metalomike u zavisnosti od bioloških i kliniĉko-patoloških parametara, kao i pronalaţenje najznaĉajnijeg metala (ili njegovog odnosa sa drugim metalom) koji bi mogao da razdvoji ispitivane grupe i obezbedi informacije o molekulskoj osnovi patofizioloških promena ispitivane bolesti tiroidne ţlezde na tkivnom nivou. Uzorci tkiva za analizu metala sakupljeni su nakon operacije tiroidne ţlezde. U istraţivanju je uĉestvovalo 200 pacijenata, od toga 21 pacijent sa Hašimotovim tiroiditisom (HT), 62 pacijenta sa koloidnom strumom (KS), 36 pacijenata sa benignim tumorima (BT) i 81 pacijent sa malignim tumorima (70 sa papilarnim karcinomom, 8 sa folikularnim i troje sa medularnim karcinomom). Od svakog pacijenta sa dijagnostifikovanim tumorom uzorkovano je i ZTT sa najveće moguće udaljenosti od primarnog tumora, koji je bio jasno demarkiran (n = 117). U svakoj patologiji ispitan je sadrţaj metala u zavisnosti od pola, uzrasta, konzumiranja duvana i veliĉine tiroidnog nodusa. Metalomika papilarnog karcinoma tiroidne ţlezde dodatno je ispitana u zavisnosti od patohistološkog (PH) tipa tumora, prethodne istorije tiroidnog oboljenja u porodici, „TNM“ klasifikacije, prisustva invazije tiroidne kapsule, intratiroidne multicentriĉne diseminacije i retrosternalnog tiroidnog rasta. Uzorci tkiva razoreni su mikrotalasnom digestijom i koncentracija elemenata, mangana (Mn), nikla (Ni), bakra (Cu), cinka (Zn), arsena (As), selena (Se), kadmijuma (Cd), olova (Pb), torijuma (Th) i uranijuma (U, The etiology of thyroid diseases is not very well clarified and the latest literature data indicated that metals could play a key role in the pathogenesis of a great number of disorders, including the thyroid gland diseases. In this regard, the aim of this study was to determine the content of toxic and essential metals in the healthy thyroid tissues (HTTs), as well as to compare the content of metals in tissues with diagnosed benign and malignant thyroid gland diseases. The further aim of this study was to investigate metallomics according to the biological-, clinical- and pathological-parameters, as well as to find the most significant metal (or its ratio to another metal) that could separate examined groups, as well as to provide additional information on the molecular basis of pathophysiological changes of the examined thyroid disease at the tissue’s level. Tissue samples for metal analysis were collected after thyroid surgery. The study included 200 patients, of which 21 patients with Hashimoto’s thyroiditis (HT), 62 patients with colloid goiter (CG), 36 patients with benign tumors (BTs) and 81 patients with malignant tumors (70 with papillary carcinoma, 8 with follicular and three with medullary thyroid carcinoma). HTT was sampled from each patient with diagnosed tumor from the greatest possible distance from the primary tumor, which was well demarked (n = 117). In each pathology, the content of metals was investigated according to sex, age, smoking habits and goiter size. Metallomics of the papillary thyroid carcinoma (PTC) was further examined based on the pathohistological (PH) type of tumor, previous history of any thyroid disease in the family, TNM classification, capsular invasion, intrathyroid dissemination, and the retrosternal thyroid growth. Tissue samples were decomposed by microwave digestion and the concentration of elements, manganese (Mn), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), lead (Pb), thorium (Th) and

Background: Antiepileptic drugs (AEDs) can cause hypersensitivity reactions in children. These reactions are mainly cutaneous, self-limiting, and benign, but life-threatening severe cutaneous adverse reactions can occur. Infections can lead to skin eruptions and mimic drug hypersensitivity reactions, if a drug is taken at the same time. The aims of our study were to confirm or rule out the diagnosis of hypersensitivity reactions to AEDs in children and to detect an infection which mimics these reactions. Methods: A prospective survey was conducted in a group of 100 children with histories of hypersensitivity reactions to AEDs by performing patch tests, delayed-reading intradermal test, and, in case of negative results, challenge test. In all children, a study was performed to detect infections by viruses or Mycoplasma pneumoniae. Results: Maculopapular exanthema and delayed-appearing urticaria were the most reported hypersensitivity reactions to AEDs. Sixty-six (66%) of 100 children had confirmed hypersensitivity reactions to AEDs. Fifty-nine children had positive patch test. No children had positive challenge tests. The most common AEDs causing hypersensitivity reactions were carbamazepine (45.4%) and lamotrigine (43.6%). Thirty-two children had positive tests for viruses or M pneumoniae, and nine of them had also a positive allergy work-up. Conclusion: Considering that there are no specific tests to distinguish between a viral infection and hypersensitivity reactions to AEDs in the acute phase, a diagnostic work-up should be performed in all children with suspected hypersensitivity reactions to AEDs, as well as infectious agent study, to remove a false label of hypersensitivity.

Background: Before introducing proteins from new or alternative dietary sources into the market, a compressive risk assessment including food allergic sensitization should be carried out in order to ensure their safety. We have recently proposed the adverse outcome pathway (AOP) concept to structure the current mechanistic understanding of the molecular and cellular pathways evidenced to drive IgE-mediated food allergies. This AOP framework offers the biological context to collect and structure existing in vitro methods and to identify missing assays to evaluate sensitizing potential of food proteins. Scope and approach: In this review, we provide a state-of-the-art overview of available in vitro approaches for assessing the sensitizing potential of food proteins, including their strengths and limitations. These approaches are structured by their potential to evaluate the molecular initiating and key events driving food sensitization. Key findings and conclusions: The application of the AOP framework offers the opportunity to anchor existing testing methods to specific building blocks of the AOP for food sensitization. In general, in vitro methods evaluating mechanisms involved in the innate immune response are easier to address than assays addressing the adaptive immune response due to the low precursor frequency of allergen-specific T and B cells. Novel ex vivo culture strategies may have the potential to become useful tools for investigating the sensitizing potential of food proteins. When applied in the context of an integrated testing strategy, the described approaches may reduce, if not replace, current animal testing approaches.

Evaluation of trace metals at level of solid tissue can provide better information than blood or urine and, therefore, could highlight the role of metals in the etiology of organ-specific disease. The current study aimed to establish the baseline content of four essential (Mn, Cu, Zn, Se) and four toxic metals (As, Cd, Pb, U) in the healthy thyroid tissues (HTTs) by considering sex, age and smoking habits. A further aim was to examine whether differences in the content of metals exist in regard to the thyroid diseases, such as benign tumor (BT), Hashimoto's thyroiditis (HT), multinodular goiter (MNG) and thyroid cancer (TC). A total number of investigated tissue samples were 423. All metals were quantified by inductively coupled plasma-mass spectrometry (ICP-MS). It was found that the content of Cu and U was higher in HTTs of women, while the content of Zn was higher in HTTs of men. Increased content of Zn and decreased content of U was found in the group of HTTs above 50 years compared to a younger group (<50 years). Increased content of Cd, Pb and U distinguish smokers from the non-smokers. In comparison with other population groups worldwide, investigated Serbian population had up to 15 times reduced content of Se. Despite the difference in metal's profile according to biological variables, this study also demonstrated, for the first time, that each thyroid disease has its unique metal's profile. The most altered metal's content was found in tissues with HT. Contrarily, the greatest similarity in metal's content with HTTs was found in BT tissues. Based on the increased content, metal's that dominantly discriminated HTTs from the HT, MNG and TC was As, Pb and Cd, respectively. Reported results could highlight the role of toxic and essential trace metals in the not very well clarified etiology of thyroid diseases and, moreover, could provide a molecular basis for pathophysiological changes of metal's hazardous effects on thyroid health at the tissue level.

Background: The etiology of papillary thyroid carcinoma (PTC) is unknown and some literature data support the hypothesis that heavy metals, as endocrine disrupters, could play a major role in the pathogenesis of thyroid cancer. This study aimed to estimate the content of selected toxic and essential trace metals (Mn, Co, Ni, Cu, Zn, As, Se, Cd, Pb, Th, and U), as well as the selected ratio's (Cu/Zn and Cd/Se) in the malignant thyroid tissues according to sex, age, smoking habits, familial history of any thyroid disease, pathohistological (PH) types of PTC, tumor size, the existence of a thyroid capsular invasion, intrathyroid tumor dissemination, retrosternal thyroid growth, and TNM progress of PTC. Methods: The study included 66 patients with PTC (women/men ratio = 46/20, mean age: 54 ± 14 years). A comparative analysis was made by collecting the healthy thyroid tissues (HTTs) of the same patients, making the total number of samples 132. All trace metals were quantified by inductively coupled plasma-mass spectrometry (ICP-MS). Results: Metals that significantly separated papillary thyroid tissues (PTTs) from the HTTs were Cd, U and Se (p < 0.05). The obtained negative correlation between Cd and Se in the PTTs could explain extrusion of essential Se caused by increased content of Cd. Only Cd had an influence on the retrosternal thyroid growth, while the essential metals (Mn, Co, and Zn) had an influence on thyroid capsular invasion. Conclusion: It was found that Cd act as the main endocrine disrupter, which could highlight its role in the etiology of PTC. Considering that the Cd/Se ratio significantly separated two studied groups and had an influence on the retrosternal thyroid growth, its altered content could contribute to the better understanding of the molecular basis for pathophysiological changes in the PTC.

Glukoza-oksidaza (GOx) je vaţan industrijski enzim koji se predominantno koristi kao biokatalizator u industriji hrane za proizvodnju glukonske kiseline, uklanjanje kiseonika i sterilizaciju. U farmaceutskoj industriji i kliničkoj biohemiji se koristi kao biosenzor za određivanje koncentracije glukoze, dok postoje pokušaji da se iskoristi i za proizvodnju biogorivnih ćelija koje proizvode električnu energiju koristeći glukozu i kiseonik iz ljudske krvi u okvirima nanobiotehnologije. Za rasprostranjeniju primenu GOx neophodno je unaprediti neke od njenih osobina kao što su aktivnost, pH optimum, reaktivnost sa kiseonikom i stabilnost. Jedna od metoda kojom se unapređuju proteini je dirigovana evolucija. Ova metoda podrazumeva iterativne tehnike generisanja biblioteka proteinskih mutanata (ili varijanti) i selekciju proteina sa odgovarajućom ţeljenom funkcijom iz ovih biblioteka.U cilju pronalaţenja mutanata GOx iz Aspergillus niger koji su oksidativno stabilniji i aktivniji, u ovom radu su razvijene dve metode pretraţivanja bazirane na ekspresiji proteina na površini ćelija kvasca. Pored razvoja metoda pretraţivanja ispitivana je i optimizacija ekspresije rekombinantne GOx u kvascu Pichia pastoris za heterolognu ekspresiju mutanata GOx i njihovu kinetičku karakterizaciju.Oksidacija bočnih ostataka amino kiselina je jedan od glavnih razloga nestabilnosti nativne trodimenzionalne strukture GOx. Posebno osetljiv prema oksidaciji je metionin, koji se prevodi u metionin-sulfoksid čak i pod blagim uslovima u prisustvu kiseonika i kiseoničnih reaktivnih vrsta kao što su vodonik-peroksid, superoksid anjon radikal i drugi. Da bi utvrdili uticajpozicije metionina na oksidativnu stabilnost GOx, svih 11 metionina je razmatrano za mesto specifičnu mutagenezu..., Glucose-oxidase (GOx) is an important industrial enzyme that is predominantly used as a biocatalyst in the food industry for the production of gluconic acid, oxygen removal and sterilization. In the pharmaceutical industry and clinical biochemistry it is used as a biosensor for determining glucose concentration, while there are attempts to utilize it in the production of biofuel cells that produce electricity using glucose and oxygen from human blood within the framework of nanobiotechnology. For the widespread use of GOx it is necessary to improve some of its properties such as activity, pH optimum, reactivity with oxygen and stability. One of the methods for improving proteins is directed evolution. This method involves iterative techniques for generating libraries of protein mutants (or variants) and selecting proteins with the desired function from these libraries.In order to find GOx mutants from Aspergillus niger that are oxidatively more stable and active, in this work two methods of screening based on protein expression on the surface of yeast cells have been developed. In addition to the development of the screening methods, the optimization of the expression of recombinant GOx in yeast Pichia pastoris for heterologous expression of GOx mutants and their kinetic characterization was studied.Aminoacid side chain oxidation is one of the main causes for structural instability of native tridimensional structure of GOx. Particularly sensitive to oxidation is methionine residue, which is converted to methionine sulfoxide even under mild conditions in the presence of oxygen and oxygen reactive species such as hydrogen peroxide, superoxide anion radical and others. In order to determine the effect of the position of the methionine amino acidresidue on the oxidative stability of GOx, all 11 methionines were considered for site directed mutagenesis...

Tetanus toksin (TT), produkt anaerobne bakterije Clostridium tetani, je izuzetnopotentan neurotoksin koji može izazvati tetanus, teško i često fatalno oboljenje. Uprkosvišedecenijskom sistematskom sprovođenju vakcinacije protiv tetanusa, incidencatetanusa još uvek nije zanemarljiva. TT-specifična antitela su ključni faktor usprečavanju intoksikacije TT-om. Pored toga, predpostavlja se da bi ova antitela, usledukrštene reaktivnosti, mogla doprinositi i otpornosti ka heterologim infekcijama. Kodosoba koja nemaju uspostavljen adekvatan TT-specifičan imunski odgovor, a postojisumnja na intoksikaciju TT-om, preporučuje se imunoterapija koja podrazumevaaplikaciju TT-specifičnih antitela sposobnih da neutrališu slobodno cirkulišući TT. Dužiniz godina se kao imunoterapeutici razmatraju i TT-specifična monoklonska antitela(MAt) gde se kao jedan od problema nameće pitanje kako selektovati zaštitno MAt i / iliformulisati efikasan preparat s obzirom da specifičnost ka TT ne znači nužno i zaštinipotencijal.Cilj ove doktorske disertacije je da se, kroz ispitivanje vezivnih karakteristikapanela TTd/TT-specifičnih MAt i njihovog zaštitnog potencijala in vivo, utvrdi na kojinačin i u kojoj meri vezivne karakteristike TTd/TT specifičnih antitela određuju njihovusposobnost da spreče intoksikaciju TT-om i / ili doprinesu otpornosti ka heterologiminfekcijama.Istraživanja realizovana u okviru izrade ove doktorske teze su pokazala da sezaštitni potencijal anti-TTd / TT antitela može proceniti na osnovu afiniteta ka TT-u isposobnosti da inhibiraju TT-GD1b interakciju. TT-specifična antitela koja mogu daspreče in vivo intoksikaciju TT-om treba da vezuju TT-om sa afinitetom ≥ 1x108 M-1 ida istovremeno imaju sposobnost da inhibiraju vezivanje TT-a za GD1b gangliozide.Afinitet prema TT-u je prvi selekcioni kriterijum zaštitnih TT-specifičnih antitela, apotom njihova sposobnost da preveniraju TT-GD1b interakciju. Zaštitni potencijal TTspecifičnihantitala čiji je afinitet ka TT-u ≥ 1x108 M-1 dom, is an extremely potent neurotoxin that can cause tetanus, a severe and often fataldisease. Mandatory vaccination against tetanus is introduced worldwide and it wascrucial for a significant decline of tetanus cases. Nevertheless, the prevalence of thedisease is not negligible, particularly in the developing world. Antibodies capable toneutralize TT are key factors in protection against tetanus disease. It is assumed thatantibodies may, due to cross-reactivity, contribute to the immunity against heterologouspathogens. TT intoxications can be efficiently treated with various polyclonal antibodybasedtherapies. Although antibody-based therapeutics for treatment of tetanus exist onthe market its production is tedious. Monoclonal antibodies (MAbs) are considered for along time as a reagent of choice, but the core drawback is how to select a MAb and howto prepare oligoclonal MAbs-based preparation that would be safe in providingefficacious protection, since available data clearly demonstrate that the ability of asingle MAb to bind TT does not necessarily mean that this MAb protects from tetanusintoxication.The aims of this thesis were to evaluate the binding characteristics andprotective capacity of TTd/TT-specific MAb(s)-based preparations, to determine howthese binding characteristics correlate with the observed in vivo effects and toinvestigate whether these preparations could contribute to the development ofheterologous immunity.In this PhD thesis, we showed that the selection of protective anti-TTd/TT MAbscan be performed by the in vitro testing combining two assays: (i) the measurement ofMAb affinity toward TT (ii) the evaluation of its capability to prevent TT-gangliosideinteraction. TT-specific antibodies that prevent in vivo TT intoxication bind TT withaffinity ≥ 1x108 M-1 and, at the same time, possess the ability to inhibit binding of TT toGD1b ganglioside. MAb’s afinity toward TT is the first selection criterion of protectiveTT-specific antibodies, and the secon

Bronchopneumonia is the most frequent cause of calves’ ailments around the world. The absence of adequate immune response to causative agents of bronchopneumonia in calves remained unclear to date. The infectious agents of virus and/or bacterium origin associated with bronchopneumonia are omnipresent harmless commensals in the cattle population that induce illness when the host defense is depressed by environmental stress factors and inability of calves to fully respond to an antigenic stimulus due to the fragile nature of the immune system. The disease is a leading cause of economic loss in the livestock industry worldwide which points to the necessity of attending to this problem with great attention. Having in mind that immune response to certain pathogens responsible for bronchopneumonia in calves is IgG antibody-dependent, the presence of immune complexes containing these antibodies could have an important role in the pathogenesis of bronchopneumonia. The goal of this doctoral dissertation was to investigate the protein and lipid components of circulating immune complexes (CIC) of calves three months old, diagnosed with bronchopneumonia caused by Pasteurella multocida bacterium, and their role as modulators of immune response. CIC were isolated by polyethylene glycol precipitation. The level of polyethylene glycol precipitating CIC in calves with bronchopneumonia compared to healthy calves was higher. In calves with bronchopneumonia CIC were larger in size. CIC were composed of five groups of corpuscles; In healthy calves the most abundant corpuscles in CIC were of 1426±291 nm in diameter; In healthy calves corpuscles with hydrodynamic radius of 225±33 nm and 734±174 nm were mostly present in CIC. In calves with bronchopneumonia zeta potential of CIC was slightly higher than zeta potential of CIC in healthy calves. In calves with bronchopneumonia increased level of serum total proteins was detected. The concentration of γ and α globulins in the serum and in CIC, Bronhopneumonija je najčešće oboljenje teladi u svetu ali do danas nije razjašnjeno zašto imunski sistem teladi ne odgovara efikasno na uzročnike bronhopneumonije. Virusi i bakterije uzročnici bronhopnemonije su komensali i izazivaju bolest samo kada je imunološka odbrana slaba, koja kod teladi nastaje zbog nezrelosti imunskog sistema i izloženosti stresu uzrokovanom nepovoljnim vremenskim prilikama i smeštajem u slabo ventilirane, prenaseljene i vlažne objekte. Kako je ovo oboljenje uzrok velikih ekonomskih gubitaka na govedarskim farmama postoji velika potreba da se problem ovog oboljenja reši. Imajući u vidu da je imunitet teleta na neke od uzročnika bronhopneumonije posredovan IgG antitelima i da bi imunokompleksi koje ova antitela formiraju mogli da učestvuju u patogenezi bronhopneumonije, ova doktorska disertacija je za cilj imala ispitivanje proteinskih i lipidnih komponenti cirkulišućih imunokompleksa (CIC) teladi starih tri meseca, obolelih od bronhopneumonije izazvane bakterijom Pasteurella multocida, najčešćim uzročnikom bolesti na farmama u okolini Beograda, i ispitivanje uloge CIC kao modulatora funkcija ćelija imunskog odgovora. U ovoj doktorskoj disertaciji CIC su izolovani precipitacijom proteina krvnog seruma polietilen glikolom. Nivo CIC teladi sa bronhopneumonijom je u odnosu na zdravu telad bio povećan. CIC teladi sa bronpneumonijom su bili veći. U CIC obe grupe teladi na osnovu hidrodinamičkog radijusa izdvaja se pet grupa čestica; u CIC bolesne teladi najzastupljenije su bile čestice od 1426±291 nm; a u CIC zdrave teladi čestice hidrodinamičkog radijusa od 225±33 nm i 734±174 nm. Zeta potencijal CIC teladi sa bronhopneumonijom je bio neznatno veći od zeta potencijala CIC zdrave teladi. U serumima teladi sa bronhopneumonijom detektovan je povišen nivo ukupnih proteina...

Tetanus toksin (TT), produkt anaerobne bakterije Clostridium tetani, je izuzetno potentan neurotoksin koji može izazvati tetanus, teško i često fatalno oboljenje. Uprkos višedecenijskom sistematskom sprovođenju vakcinacije protiv tetanusa, incidenca tetanusa još uvek nije zanemarljiva. TT-specifična antitela su ključni faktor u sprečavanju intoksikacije TT-om. Pored toga, predpostavlja se da bi ova antitela, usled ukrštene reaktivnosti, mogla doprinositi i otpornosti ka heterologim infekcijama. Kod osoba koja nemaju uspostavljen adekvatan TT-specifičan imunski odgovor, a postoji sumnja na intoksikaciju TT-om, preporučuje se imunoterapija koja podrazumeva aplikaciju TT-specifičnih antitela sposobnih da neutrališu slobodno cirkulišući TT. Duži niz godina se kao imunoterapeutici razmatraju i TT-specifična monoklonska antitela (MAt) gde se kao jedan od problema nameće pitanje kako selektovati zaštitno MAt i / ili formulisati efikasan preparat s obzirom da specifičnost ka TT ne znači nužno i zaštini potencijal. Cilj ove doktorske disertacije je da se, kroz ispitivanje vezivnih karakteristika panela TTd/TT-specifičnih MAt i njihovog zaštitnog potencijala in vivo, utvrdi na koji način i u kojoj meri vezivne karakteristike TTd/TT specifičnih antitela određuju njihovu sposobnost da spreče intoksikaciju TT-om i / ili doprinesu otpornosti ka heterologim infekcijama. Istraživanja realizovana u okviru izrade ove doktorske teze su pokazala da se zaštitni potencijal anti-TTd / TT antitela može proceniti na osnovu afiniteta ka TT-u i sposobnosti da inhibiraju TT-GD1b interakciju. TT-specifična antitela koja mogu da spreče in vivo intoksikaciju TT-om treba da vezuju TT-om sa afinitetom ≥ 1x108 M-1 i da istovremeno imaju sposobnost da inhibiraju vezivanje TT-a za GD1b gangliozide. Afinitet prema TT-u je prvi selekcioni kriterijum zaštitnih TT-specifičnih antitela, a potom njihova sposobnost da preveniraju TT-GD1b interakciju. Zaštitni potencijal TTspecifičnih antitala čiji je afinite, is an extremely potent neurotoxin that can cause tetanus, a severe and often fatal disease. Mandatory vaccination against tetanus is introduced worldwide and it was crucial for a significant decline of tetanus cases. Nevertheless, the prevalence of the disease is not negligible, particularly in the developing world. Antibodies capable to neutralize TT are key factors in protection against tetanus disease. It is assumed that antibodies may, due to cross-reactivity, contribute to the immunity against heterologous pathogens. TT intoxications can be efficiently treated with various polyclonal antibodybased therapies. Although antibody-based therapeutics for treatment of tetanus exist on the market its production is tedious. Monoclonal antibodies (MAbs) are considered for a long time as a reagent of choice, but the core drawback is how to select a MAb and how to prepare oligoclonal MAbs-based preparation that would be safe in providing efficacious protection, since available data clearly demonstrate that the ability of a single MAb to bind TT does not necessarily mean that this MAb protects from tetanus intoxication. The aims of this thesis were to evaluate the binding characteristics and protective capacity of TTd/TT-specific MAb(s)-based preparations, to determine how these binding characteristics correlate with the observed in vivo effects and to investigate whether these preparations could contribute to the development of heterologous immunity. In this PhD thesis, we showed that the selection of protective anti-TTd/TT MAbs can be performed by the in vitro testing combining two assays: (i) the measurement of MAb affinity toward TT (ii) the evaluation of its capability to prevent TT-ganglioside interaction. TT-specific antibodies that prevent in vivo TT intoxication bind TT with affinity ≥ 1x108 M-1 and, at the same time, possess the ability to inhibit binding of TT to GD1b ganglioside. MAb’s afinity toward TT is the first selection criterion of protective TT-specific

Aim To investigate the immunomodulatory potential of a chimera composed of the receptor-binding domain of hemagglutinin 1 (H1s) from Influenza virus and Der p 2 (D2) allergen for allergen-specific immunotherapy of house-dust mite allergy (HDM). Main methods: H1sD2 chimera and D2 allergen were produced by genetic engineering in E. coli. Recombinant antigens were extracted from inclusion bodies by urea, then refolded and purified by immobilized-metal affinity chromatography (IMAC). Purity was verified by 2D-PAGE and secondary structures were assessed by CD spectroscopy. IgE reactivity of H1sD2 and D2 was tested in western blot with sera from 8 persons with clinical history of HDM allergy. Immunogenicity of H1sD2 and D2 were analyzed in Balb/c mice. Cytokine profile was analyzed by ELISA after stimulation of mouse spleen cells with H1sD2 and D2. Leukocyte population abundance of cells isolated from spleen and lymph node was assessed by flow cytometry. Key findings: Purified recombinant proteins H1sD2 (42 kDa) and D2 (15 kDa) revealed well defined secondary structures, and preserved IgE reactive epitopes. Analysis of supernatants of mouse spleen cells after stimulation with H1sD2 and D2, revealed a qualitatively different cytokine profile from H1sD2 immunized mouse cells (increase in IL10). CD8+ cells were decreased in the lymph node of D2 immunized mice, whereas H1sD2 immunization led to an increase of CD8+ cells in both the lymph node and the spleen. Significance: H1sD2 chimera attenuates Der p 2-inherent Th2 response and directs the immune response toward Th1 and Treg phenotype.

Бронхопнеумонија је најчешће обољење телади у свету али до данас нијеразјашњено зашто имунски систем телади не одговара ефикасно на узрочникебронхопнеумоније. Вируси и бактерије узрочници бронхопнемоније сукоменсали и изазивају болест само када је имунолошка одбрана слаба, која кодтелади настаје због незрелости имунског система и изложености стресуузрокованом неповољним временским приликама и смештајем у слабовентилиране, пренасељене и влажне објекте. Како је ово обољење узрок великихекономских губитака на говедарским фармама постоји велика потреба да сепроблем овог обољења реши.Имајући у виду да је имунитет телета на неке од узрочникабронхопнеумоније посредован IgG антителима и да би имунокомплекси које оваантитела формирају могли да учествују у патогенези бронхопнеумоније, овадокторска дисертација је за циљ имала испитивање протеинских и липиднихкомпоненти циркулишућих имунокомплекса (CIC) телади старих три месеца,оболелих од бронхопнеумонијe изазване бактеријом Pasteurella multocida,најчешћим узрочником болести на фармама у околини Београда, и испитивањеулоге CIC као модулатора функција ћелија имунског одговора.У овој докторској дисертацији CIC су изоловани преципитацијомпротеина крвног серума полиетилен гликолом. Ниво CIC телади сабронхопнеумонијом је у односу на здраву телад био повећан. CIC телади сабронпнеумонијом су били већи. У CIC обе групе телади на основухидродинамичког радијуса издваја се пет група честица; у CIC болесне теладинајзаступљеније су биле честице од 1426±291 nm; а у CIC здраве телади честицехидродинамичког радијуса од 225±33 nm и 734±174 nm. Зета потенцијал CICтелади са бронхопнеумонијом је био незнатно већи од зета потенцијала CICздраве телади.У серумима телади са бронхопнеумонијом детектован је повишен нивоукупних протеина..., Bronchopneumonia is the most frequent cause of calves’ ailments around theworld. The absence of adequate immune response to causative agents ofbronchopneumonia in calves remained unclear to date. The infectious agents of virusand/or bacterium origin associated with bronchopneumonia are omnipresent harmlesscommensals in the cattle population that induce illness when the host defense isdepressed by environmental stress factors and inability of calves to fully respond to anantigenic stimulus due to the fragile nature of the immune system. The disease is aleading cause of economic loss in the livestock industry worldwide which points to thenecessity of attending to this problem with great attention.Having in mind that immune response to certain pathogens responsible forbronchopneumonia in calves is IgG antibody-dependent, the presence of immunecomplexes containing these antibodies could have an important role in the pathogenesisof bronchopneumonia. The goal of this doctoral dissertation was to investigate theprotein and lipid components of circulating immune complexes (CIC) of calves threemonths old, diagnosed with bronchopneumonia caused by Pasteurella multocidabacterium, and their role as modulators of immune response.CIC were isolated by polyethylene glycol precipitation. The level ofpolyethylene glycol precipitating CIC in calves with bronchopneumonia compared tohealthy calves was higher. In calves with bronchopneumonia CIC were larger in size.CIC were composed of five groups of corpuscles; In healthy calves the most abundantcorpuscles in CIC were of 1426±291 nm in diameter; In healthy calves corpuscles withhydrodynamic radius of 225±33 nm and 734±174 nm were mostly present in CIC. Incalves with bronchopneumonia zeta potential of CIC was slightly higher than zetapotential of CIC in healthy calves.In calves with bronchopneumonia increased level of serum total proteins wasdetected. The concentration of γ and α globulins in the serum and in CIC of diseasedcalves was hi

Diagnostic reagents based on food allergen extracts often lack sufficient sensitivity. The introduction of well characterized food allergens in molecular allergy diagnosis has been recognized as valid approach to circumvent unstandardized allergen extracts. Banana fruit (Musa acuminate) is a well-established allergen source which besides six characterized allergens, contains unidentified IgE reactive proteins whose clinical relevance remains undefined. By employment of a combinatorial peptide ligand library (CPLL) methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis, a novel allergen from banana fruit was detected in banana as catalase. A recombinant homologue of natural catalase was produced, isolated and biochemically characterized. The recombinant protein showed IgE reactivity in 7 out of 13 tested patients with suspected allergy to banana in immunoblot. Novel banana fruit allergens should be added as components to allergen-microarrays for the diagnosis and the monitoring of banana allergy. Significance: By employment of CPLL methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis catalase from banana fruit is identified as a novel allergen, with proposed designation as Mus a 7. IgE reactive recombinant Mus a 7 was produced and should be included in a component-resolved allergy diagnosis.

Složeni ugljeni hidrati kao što je skrob, od velikog su industrijskog značaja kako uoblastima bioenergije i bioprocesovanja, tako i u ljudskoj ishrani. Enzimi koji mogu direktnoda hidrolizuju sirovi (nativni, granularni) skrob na temperaturama nižim od temperaturegeliranja privlače sve veći interes jer bi njihovom upotrebom mogao da se smanji utrošakvelike količine energije u zagrevanju skroba prilikom geliranja, a time i pojeftini proces.Rekombinantna α-amilaza iz Bacillus licheniformis ATCC 9945a (BliAmy), efikasna uhidrolizi granularnog skroba ispod temperature geliranja, proizvedena je ekstracelularnokorišćenjem Escherichia coli kao domaćina. Razvijeni su efikasni i reproduktivni procesifermentacije sa ćelijama E. coli BL21 (DE3) i E. coli C43 (DE3) kroz dizajn medijuma zarast mikroorganizma i optimizaciju koraka u proizvodnji amilaze. Dobijene su velike količineBliAmy i to, u fermentaciji sa ćelijama E. coli BL21 (DE3) ukupna amilazna aktivnost od250,5 IU mL-1 (0,7 g L-1 ), a sa ćelijama E. coli C43 (DE3) 500 IU mL−1 (1,2 g L−1).Primenjen je eksperimentalni dizajn u optimizaciji hidrolize skroba, prečišćavanjuproteina i dobijanja bioetanola. Korišćenjem statističke metode odgovornih površinaoptimizovan je proces hidrolize koncentrovane suspenzije sirovog kukuruznog skroba.Pokazano je da je BliAmy veoma efikasna jer je hidrolizovala 91 % prilikom hidrolize 30 % -ne suspenzije skroba nakon 24 sata.Na osnovu promena u kristalnoj strukturi analiziranjem rezidualnog skroba kojizaostaje nakon hidrolize 30%-ne susupenzije nakon 5 i 24 sata pokazano je da kukuruzniskrob ima A-tip i Vh-tip kristaliniteta. Upoređivanjem difrakcionih intenziteta pikovakristalne strukture i SEM mikrografije nativnog i rezidualnog skroba može se zaključiti da seistom brzinom hidrolizuju i kristalni i amorfni regione u granulama skroba, što je u skladu sanačinom hidrolize skrobnih granula tzv. „granula po granula“..., Complex carbohydrates such as starch are of great industrial interest, especially inthe areas of bioenergy and bioprocessing and increasingly in human nutrition. Theimportance of enzymes capable of direct hydrolysis of raw (native, granular) starch belowthe temperature of gelatinization has been well recognized due to the energy savings andthe effective utilization of biomass, which reduces the overall cost of starch processing.Recombinant α-amylase from Bacillus licheniformis ATCC 9945a (BliAmy), highlyefficient in raw starch hzdrolysis was produced extracellularly in Escherichia coli. Anefficient and reproducible fermentation approach was developed with cells E. coli BL21(DE3) and E. coli C43 (DE3) through the smart design of defined growth medium andoptimization of process steps. Significant overexpression of BliAmy was achieved. UsingE. coli BL21 (DE3) total of 250.5 IU mL-1 (0.7 g L-1 ) was obtained while using E. coliC43 (DE3) total of 500 IU mL−1 (1.2 g L−1) was achived.Experimental design was applied to optimize the hydrolysis of starch, proteinpurification and production of bioethanol. The hydrolysis of concentrated raw starch wasoptimized using response surface methodology. BliAmy was very effective, achieving thefinal hydrolysis degree of 91 % for the hydrolysis of 30 % starch suspension after 24 h.Cystalline structure of starch residues analysed by X-ray diffraction on the samplesoriginating from 30 % starch suspensions after 5 and 24 h shown A-type and a Vh-typecrystallinity of corn starch. Constant diffracted intensities and scattering backgroundindicated that BliAmy most likely degraded both amorphous and crystalline areas at thesamerates. This is in agreement with a granule by granule mode of attack and can be concludedfrom the SEM micrographies...

Peroksidaza iz soje (SBP, E.C. 1.11.1.7) je enzim koji pripada klasi III biljnih peroksidaza i katalizuje oksidaciju i polimerizaciju različitih fenolnih jedinjenja u prisustvu vodonik peroksida kao supstrata. Nalazi se u semenom omotaču soje koji predstavlja jeftini nusproizvod i polazni materijal za izolovanje velike količine sirovog enzima.Izolovana SBP je imobilizovana primenom dve kovalentne metode na seriju makroporoznih kopolimera poli(GMA-co-EGDMA) različitih dijametara pora. Glutaraldehidni metod imobilizacije se pokazao boljim od perjodatnog a najveća specifična aktivnost od 23 IU/g dobijena je na kopolimeru veličine pora od 120 nm. Imobilizovana peroksidaza pokazala je veću termalnu stabilnost i stabilnost u organskom rastvaraču, aktivnost u širem opsegu pH i 2,86 puta veću Km vrednost za pirogalol u odnosu na rastvorni enzim.Sintetisana je serija modifikovanih pektina oksidacijom perjodatom u molarnom odnosu od 2,5 do 20 mol% i naknadnom reduktivnom aminacijom tiraminom i cijanoborhidridom. Dobijeni tiramin-pektini prave stabilne hidrogelove umrežavanjem fenolnih grupa u reakciji katalizovanoj peroksidazama u prisustvu vodonik peroksida. SBP je uspešno imobilizovana unutar mikro-kuglica hidrogela nastalih enzimskom polimerizacijom modifikovanog pektina u emulziji sa najvećom specifičnom aktivnošću postignutom na tiramin-pektinu oksidovanom 5 mol% perjodatom. Imobilizovana SBP je pored šireg pH optimuma pokazala i veću termalnu stabilnost i stabilnost u organskom rastvaraču u odnosu na slobodan enzim. Mikrokuglice sa imobilizovanom SBP zadržale su 50% početne aktivnosti nakon 7 ciklusa ponovne upotrebe za oksidaciju pirogalola u bač reaktoru.Pod optimalnim uslovima sa unutrašnjom dostavom vodonik peroksida postignuto je uklanjanje fenola od 64% imobilizovanom SBP na poli(GMA-co-EGDMA) veličine pora od 120 nm., Soybean peroxidase (SBP, E.C. 1.11.1.7) is an enzyme that belongs to class III of plant peroxidases that catalyses oxidation and polymerization of various phenolic compounds in the presence of hydrogen peroxide as substrate. It's located in the soybean seed hulls, an inexpensive byproduct and excellent material for isolation of large amounts of crude enzyme.SBP was immobilized by using two covalent methods onto a series of macroporous copolymers of poly(GMA-co-EGDMA) with various pore diameters. Glutaraldehyde immobilization method proved to be better than periodate with highest specific activity of 23 IU/g obtained with copolymer with pore size of 120 nm. Compared to the soluble enzyme, immobilized peroxidase showed increased thermal and organic solvent stability, broader pH activity range and 2.86 times higher Km value for pyrogallol.Series of modified pectins have been synthesized by oxidation with periodate in molar ratios from 2.5 to 20 mol% and subsequent reductive amination with tyramine and cyanobohrhydride. Obtained tyramnine-pectine made stable hydrogels by crosslinking phenol groups in a peroxidase catalysed reaction in the presence of hydrogen peroxide. SBP was immobilized within hydrogel microbeads created in enzyme polymerization of modified pectin in emulsion with highest specific activity achieved with tyramine-pectin oxidized by 5 mol% of periodate. Immobilized SBP in addition of having wider pH optimum showed higher thermal and organic solvent stability compared to the free enzyme. SBP bound in microbeads retained 50% of the original activity after 7 cycles of repeated usage for pyrogallol oxidation in batch reactor.Under optimal conditions with internal hydrogen peroxide delivery phenol removal of 64% has been achieved with SBP immobilized onto poly(GMA-co-EGDMA) with pore size of 120 nm.