Immunological characterization of the linden pollens and modulation of the immune response by naturally occurring aromatic compounds

Učestalost pojave alergijskog sindroma u poslednjih par decenija je značajno uvećana, te se alergije i alergijska oboljenja danas često nazivaju „bolest 21. veka“. Prema trenutnim podacima, oko 25% svetske populacije pati od nekog oblika alergije, dok u ekonomski razvijenim zemljama taj procenat seže i do 40%. Postoji nekoliko hipoteza koje pokušavaju da objasne ovaj trend porasta učestalosti alergijske reakcije, ali uzrok još uvek nije poznat. Alergijska reakcija se javlja kada IgE antitela alergične osobe prepoznaju specifične proteine iz polena, tzv. alergene. Poleni drveća, trava i korovskih biljaka smatraju se jednim od najčešćih izvora alergena, te samim tim i izazivačima alergijske reakcije. Alergeni potencijal specifičnog polena ne zavisi samo od alergične osobe, već i od godišnjeg doba i geografskog regiona. Tako, za predeo severne Evrope karakteristična je alergija na polen breze (Betula verrucosa), dok je u predelu južne Evrope i Mediterana prisutna alergija na polen masline (Olea europea). Međutim, pored ovih poznatih izvora alergena, nije izučeno da li poleni drugih široko raspostranjenih biljnih vrsta izazivaju alergijske reakcije. U ovoj doktorskoj disertaciji je izučavan alergijski odgovor na polen korišćenjem dva model sistema. U prvom je izučavana alergija humane populacije na polen lipe, dok je u drugom izučavan alergijski odgovor populacije kućnih pasa na polen kratke ambrozije. U ovoj doktorskoj disertaciji je, po našem saznanju, po prvi put urađena karakterizacija alergenog potencijala polena lipe u humanoj populaciji. Takođe, u ovom radu prikazana je analiza ukrštene reaktivnosti polena lipe sa polenima masline i breze u humanim populacijama iz različitih geografskih i klimatskih regiona. Naši rezultati ukazuju da je lipa potencijalni izvor alergena, da postoji visok stepen ukrštene reaktivnosti sa polenima drugih vrsta drveća i da je potrebno uvrstiti ovaj polen u kliničku paletu polena koja se koristi u alergološkoj dijagnostici. Takođe, u ovoj disertaciji potvrđeno je da polen kratke ambrozije u Srbiji uzrokuje atopični dermatitis kod populacije kućnih pasa, određeni su njegovi glavni alergeni izazivači, urađena je uporedna analiza profila alergijskih reakcija pasa i humanih pacijenata na polen ambrozije, i ustanovljene sličnosti i razlike u karakteru i identitetu glavnih alergena...The prevalence of allergic reactions has increased significantly in the last few decades, which prompted the scientific community to name allergies and allergic disorders as “the disease of the 21st century”. According to the current data, approximately 25% of the world population suffers from some form of allergy, whereas this number approaches 40% in the Western industrialized countries. Several hypotheses had been put forward in an attempt to explain the basis for the increase in the allergy prevalence, yet the cause remains unknown. An allergic reaction appears when the IgE antibodies of the allergic individual recognize specific proteins from the pollen, which are called allergens. Perhaps the most frequent sources of allergens and causes of allergies are the pollens from trees, grasses and weeds. The allergenic potential of the particular pollen depends not only on the allergic individual, but also on the season and geographic region. For instance, allergy against the birch pollen (Betula verrucosa) is present in the Northern Europe, whereas the allergic reaction to the olive tree pollen (Olea europea) is widespread in the Southern Europe and Mediterranean. However, besides the well-known sources of allergens, it is not clear if pollens from other plant species that are well distributed across the continent are capable of eliciting the allergic response. In this doctoral thesis, the allergic response to pollen was studied by using two model systems. In one, the allergic response of human patients against the linden pollen was characterized, while in the other, the canine allergy in Serbia against the pollen from short ragweed was studied. Herein, a detailed characterization of the allergenic potential of the linden pollen among human patients is presented for the first time. Also, results supporting the crossreactivity between the pollens from the linden, birch, and olive trees in patients from distinct climate and geographic regions in Europe are shown. The findings suggest that the linden tree is a potential source of allergens, that there is a significant level of crossreactivity between the linden tree pollen and the pollens from other tree species, and that it is advisable to incorporate the linden pollen into the clinical pallete of allergens..

Izolovanje funkcionalne ukupne RNK iz lišća i polena lipe (Tilia cordata)

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol (TM) preparation of the total RNA. The total RNA isolated using TRIzol (TM) was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first-and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8 +/- 7.9 mu g g(-1) leaves; 25.7 +/- 1.1 mu g g(-1) pollen) could be obtained and was suitable for application in further molecular biology studies.Uspostavljeni su uslovi za izolovanje ukupne RNK iz lišća i polena evropske lipe (Tilia cordata). Korišćenjem komercijalno dostupnog pribora za izolovanje RNK iz biljaka izolovana je čista ukupna RNK iz lišća lipe, dok je korišćenjem iste metode dobijena degradirana RNK iz polena lipe. Uspešno izolovanje RNK iz lišća i polena je dobijeno korišćenjem TRIzol reagensa. RNK izolovana ovim metodom je kontaminirana genomskom DNK, koja je uspešno eliminisana korišćenjem enzima DNaze. Dalje su optimizovani i uslovi uklanjanja genomske DNK pomoću DNaze. Izolovana ukupna RNK iz oba izvora je dalje uspešno iskorišćena za sintezu prvog i drugog lanca klonske DNK, kao i u reverzno-transkriptivnoj PCR reakciji, dokazujući time da je korišćenjem ovog metoda izolovana funkcionalna ukupna RNK. U zaključku, dobijena je čista i funkcionalna RNK iz lišća i polena T. cordata (27,8±7,9 μg g-1 lišća; 25,7±1,1 μg g-1 polena) koja se može koristiti u daljim molekularno-biološkim istraživanjima

Evaluacija kriterijuma za dijagnozu atopijskog dermatitisa i detekcija alergen specifičnih IgE antitela kod pasa alergičnih na polen biljke Ambrosia artemisiifolia

Common ragweed (Ambrosia atremisiifolia) is one of the most frequent causes of pollen-induced allergic reactions both in humans and dogs. It has not been defined yet, what is the major allergen(s) to which most dogs allergic to ragweed show a positive result on intradermal skin test (IDST). In the present study sensitization to Ambrosia artemisiifolia pollen allergens in dogs with atopic dermatitis was examined with both in vivo and in vitro tests, including IDST and serum allergen specific IgE test. Detection of specific-IgE antibodies against ragweed allergens by immunoblotting in the sera of allergic dogs was optimized, as well. Dogs that were positive, as judged by IDST reactions to ragweed pollen allergens, also had alergen specific IgE antibodies in their sera. Results indicate that major allergens of A. artemisifolia pollen in dogs are Amb a 1 and Amb a 2. Further characterization of ragweed allergens is needed before they could potentially be used in intradermal testing or allergen immunotherapy in affected dogs. Also, we evaluated new Favrots diagnostic criteria for canine atopic dermatitis in dogs allergic to Ambrosia atremisiifolia pollen. It might be concluded that proposed criteria are of great assistance for seting up suspected diagnosis of canine atopic dermatitis, after ruling out other pruritic dermatoses.Kratka ambrozija (Ambrosia artemisiifolia) je jedan od najčešćih uzročnika alergijskih reakcija izazvanih polenom kod ljudi i pasa. Još uvek nije definisano koji je glavni alergen (i), na koji, većina pasa alergičnih na polen ambrozije, ispoljava pozitivnu reakciju na intradermalnom testu. U ovoj studiji je ispitana senzibilizacija na polen ove biljke kod pasa sa simptomima atopijskog dermatitisa in vivo i in vitro testovima, uključujući intradermalni test i dokazivanje prisustva alergen specifičnih IgE antitela u serumu. Optimizovani su uslovi za detekciju IgE specifičnih antitela iz seruma pasa alergičnih na polen ambrozije imunoblot tehnikom. Psi koji su imali pozitivnu reakciju na polen ove biljke na intradermalnom testu, takođe su imali specifična IgE antitela u serumu. Dobijeni rezultati ukazuju da su glavni alergeni Ambrosia artemisiifolia kod pasa Amb a 1 i Amb a 2. Neophodna je dalja karakterizacija alergena ambrozije kako bi se oni mogli primeniti pri rutinskom intradermalnom testiranju ili u alergen specifičnoj imunoterapiji obolelih pasa. Takođe je razmatrana i validnost Favrotovih dijagnostičkih kriterijuma kod pasa alergičnih na polen ambrozije. Može se zaključiti da su predloženi kriterijumi od velike pomoći u postavljanju suspektne dijagnoze atopijskog dermatitisa pasa, nakon isključenja drugih pruritičnih dermatoza

Digestibilnost β-laktoglobulina nakon njegovog unakrsnog povezivanja dejstvom lakaze iz Trametes versicolor i polifenola iz jabuke

beta-Lactoglobulin (BLG) is an important nutrient of dairy products and an important allergen in cow's milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE) and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG), with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders.β-Laktoglobulin (BLG) je važan nutrijent mlečnih proizvoda i važan alergen kod alergija na kravlje mleko. Cilj ove studije je bilo ispitivanje potencijala lakaze da unakrsno poveže BLG u prisustvu fenolnog ekstrakta jabuke (APE), kao i karakterizacija dobijenih proizvoda sa aspekta njihove digestibilnosti pepsinom i pankreatinom. Kompozicija fenola jabuke korišćenih za unakrsno povezivanje određena je pomoću LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne količine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri čemu je polimerizacija učinila BLG nerastvornim u reakcionoj smeši. SDS-PAGE analiza pokazala je da reakciona smeša sadrži heterogenu smešu velikih molekulskih masa (unakrsno povezan BLG), kao i deo zaostalog monomernog BLG. Enzimsko procesovanje BLG lakazom, u prisustvu polifenola jabuke kao medijatora, može smanjiti bifaznu pepsin-pankreatinsku digestibilnost kako monomernog, tako i unakrsno povezanog BLG, i na taj način smanjiti njegovu nutritivnu vrednost. Takođe, smanjena digestibilnost BLG može smanjiti njegov alergeni potencijal. Polifenoli jabuke mogu se koristiti za kreiranje novih, funkcionalnijih prehrambenih proizvoda, dizajniranih za prevenciju kako gojaznosti, tako i bolesti vezanih za preosetljivost

Supplementary data for the article: Stojadinovic, M.; Radosavljevic, J.; Ognjenovic, J.; Vesic, J.; Prodic, I.; Stanic-Vucinic, D.; Cirkovic Velickovic, T. Binding Affinity between Dietary Polyphenols and β-Lactoglobulin Negatively Correlates with the Protein Susceptibility to Digestion and Total Antioxidant Activity of Complexes Formed. Food Chemistry 2013, 136 (3–4), 1263–1271. https://doi.org/10.1016/j.foodchem.2012.09.040

Supplementary material for: [https://doi.org/10.1016/j.foodchem.2012.09.040]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1577

Isolation of functional total RNA from Tilia cordata leaves and pollen

Conditions for isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing Qiagen plant mini kit while total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIZOL™ preparation of total RNA. Total RNA isolated using TRIZOL™ was contaminated with genomic DNA but treatment with enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, conditions for elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. Isolated total RNA from both leaves and pollen was used successfully in firstand second-strand cDNA synthesis reactions, as well as, in RT-PCR, demonstrating that the total RNA isolated using this method is functional. In conclusion, pure and functional total RNA from Tilia cordata leaves and pollen (27.8 ± 7.9μg/g leaves; 25.7 ± 1.1μg/g pollen) can be obtained and applicable for further molecular biology studies

Digestibility and allergenicity of beta-lactoglobulin following laccase-mediated cross-linking in the presence of sour cherry phenolics

beta-Lactoglobulin (BLG) is an important nutrient of dairy products, but it represents a serious health risk in patients allergic to milk. Sour cherry (Prunus cerasus L) extract (SCE) is frequently added as a natural food colour in composite foods, such as fruit yogurt, ice creams, frappes and milkshakes. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an SCE and to characterise the obtained products for their bioactivity. Laccase cross-linked BLG in the presence of sour cherry phenolics. In a basophil-activation assay, the allergenicity of the cross-linked protein was shown to decrease in all nine cow's milk-allergic patients, while digestibility of the remaining monomeric BLG in simulated conditions of the gastrointestinal tract increased. Tryptic peptides became immediately available in BLG treated by laccase and laccase/SCE. The hydrolysates obtained by trypsin digestion of BLG/laccase/SCE showed an increase of 57% in radical-scavenging activity, compared to the control BLG. Enzymatic processing and usage of natural phenolic extracts as mediators of enzymatic reaction may improve BLG safety and the availability of peptides following digestion, while conserving its bioactivity

Binding affinity between dietary polyphenols and beta-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed

Non-covalent interactions between beta-lactoglobulin (BLG) and polyphenol extracts of teas, coffee and cocoa were studied by fluorescence and CD spectroscopy at pH values of the gastrointestinal tract (GIT). The biological implications of non-covalent binding of polyphenols to BLG were investigated by in vitro pepsin and pancreatin digestibility assay and ABTS radical scavenging activity of complexes formed. The polyphenol-BLG systems were stable at pH values of the GIT. The most profound effect of pH on binding affinity was observed for polyphenol extracts rich in phenolic acids. Stronger non-covalent interactions delayed pepsin and pancreatin digestion of BLG and induced beta-sheet to alpha-helix transition at neutral pH. All polyphenols tested protected protein secondary structure at an extremely acidic pH of 1.2. A positive correlation was found between the strength of protein-polyphenol interactions and (a) half time of protein decay in gastric conditions (R-2 = 0.85), (b) masking of total antioxidant capacity of protein-polyphenol complexes (R-2 = 0.95).Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2905

Immunoproteomic characterization of Ambrosia artemisiifolia pollen allergens in canine atopic dermatitis

Canine atopic dermatitis (CAD) is an immune system disorder that affects 10-15% of the canine population. Short ragweed (Ambrosia artemisiifolia) pollen represents one of the major seasonal sources of allergenic pollen proteins in Europe, particularly in the Pannonian valley of the Balkan region. In Serbia, about 66% of atopic dogs showed a positive intradermal skin test with its pollen extract, which is second to house dust mites. Therefore, characterization of Ambrosia artemisiifolia pollen components, in terms of defining major and minor allergens that induce clinically manifested allergic reaction in dogs, is important for valid diagnosis and efficient therapy. This study has, for the first time, characterized and identified major Ambrosia artemisiifolia allergens in CAD, using an immunoproteomic approach. To assess the prevalence of specific IgE in electrophoretically separated ragweed pollen proteins, individual reactivity of sera from dogs with CAD was analyzed and compared to the reactivity of sera from healthy dogs in the non-reducing conditions, which were found optimal for specific canine IgE detection. A specific IgE band (38 kDa) was recognized as the most dominant allergen in CAD, occurring in 81% of positive dog's sera. 2-D immunoblotting followed by a mass spectrometry peptide fingerprint analyses with pooled canine and human atopic sera, revealed that 38 kDa major Ambrosia atremisiifolia allergens in CAD were all five isoallergens of the Amb a 1 group (antigen E), including the previously named Amb a 2 (antigen K). In contrast to canine sera, human atopic sera also recognized lower mass allergens such as the 13 fragment of Amb a 1 and profilins (Amb a 8 variants). The most prominent ragweed proteins in CAD, represent, as in humans, variants of all five isoallergens of the Amb a 1 group (pectate lyase): Amb a 1.0101 and its natural variant E1XUL2, Amb a 1.0202, 1.0304, 1.0402 and the natural variant of Amb a 1.0501, E1XUM0, as well as the a fragment of pollen allergen Amb a 1.0201. (C) 2013 Elsevier B.V. All rights reserved