79 research outputs found
Supplementary data for article: GrozdanoviÄ, M. M.; Burazer, L. M.; GavroviÄ-JankuloviÄ, M. Kiwifruit (Actinidia Deliciosa) Extract Shows Potential as a Low-Cost and Efficient Milk-Clotting Agent. International Dairy Journal 2013, 32 (1), 46ā52. https://doi.org/10.1016/j.idairyj.2013.03.001
Supplementary material for: [https://doi.org/10.1016/j.idairyj.2013.03.001]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1589
Ispitivanje stabilnosti vakcine kuÄne praÅ”ine za sublingvalnu imunoterapiju
Allergen-specific immunotherapy with house dust mite (HDM) allergen extracts can effectively alleviate the symptoms of allergic rhinitis and asthma. The efficacy of the immunotherapeutic treatment is highly dependent on the quality of house dust mite vaccines. This study was performed to assess the stability of house dust mite allergen vaccines prepared for sublingual immunotherapy. Lyophilized Dermatophagoides pteronyssinus (Dpt) mite bodies were the starting material for the production of sublingual vaccines in four therapeutic concentrations. The stability of the extract for vaccine production, which was stored below 4 Ā°C for one month, showed consistence in the protein profile in SDS PAGE. ELISA-inhibition showed that the potencies of Dpt vaccines during a 12 month period were to 65-80 % preserved at all analyzed therapeutic concentrations. This study showed that glycerinated Dpt vaccines stored at 4Ā°C preserved their IgE-binding potential during a 12 month period, implying their suitability for sublingual immunotherapeutic treatment of HDM allergy.Alergen-specifiÄna imunoterapija predstavlja postupak koji može da promeni tok bolesti kod alergijskog rinitisa i astme. Kvalitet vakcine pripremljene od kuÄnih grinja znaÄajno utiÄe na efikasnost imunoterapeutskog tretmana. Ispitivanje je imalo za cilj procenu stabilnosti vakcine kuÄnih grinja namenjene za sublingvalnu imunoterapiju. Telo liofilozovanih Dermatophagoides pteronyssinus grinja (Dpt-grinja) upotrebljeno je kao polazni materijal za proizvodnju sublingvalne vakcine u 4 razliÄita terapeutska razblaženja. Potentnost Dpt vakcina praÄena ELISA-inhibicijom u 4 terapeutska razblaženja, nakon 12 meseci zadržan je u svim razblaženjima u opsegu 65-80%. Ispitivanje je pokazalo da glicerolom stabilizovane Dpt vakcine za subligvalnu imunoterapiju, uz Äuvanje na 4Ā°C zadržavaju alergeni potencijal ( gt 65%) nakon 12 meseci od njihove pripreme, i kao takve mogu se koristiti za tretman alergije na kuÄne grinje
Hemijske modifikacije Art v 1, glavnog alergena Artemisia vulgaris, cis-akonitilovanjem i citrakonilovanjem
Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen, a significant cause of hay fever all over Europe. Specific immunotherapy is the only treatment modality for allergic disease. Application of modified allergens makes the treatment safer and more efficient. In this work, two out of three (citraconic anhydride, cis-aconitic anhydride, 2,3-dimethylmaleic anhydride) tested anhydrides were proven to be suitable for chemical modifications of allergens. Art v 1 was modified by cis-aconitylation and citraconylation in order to obtain derivatives of Art v 1 that may be suitable for further immunological testing. Acylation of Art v 1 gave derivatives (caaArt v 1 and citArt v 1) with about 80 % modified amino groups. The derivatives were in the monomeric form and had dramatically reduced pI values. Both derivatives were relatively stable at neutral pH values, while the acyl groups undergo hydrolysis under acidic conditions. Modification of allergens by cis-aconitylation and citraconylation could be a new tool for obtaining allergoids.Art v1 je glavni alergen polena crnog pelina (Artemisia vulgaris), znaÄajnog uzroÄnika polenske groznice Å”irom Evrope. Alergen-specifiÄna imunoterapija je za sada jedini delotvoran naÄin za tretiranje alergija, pri Äemu primena modifikovanih alergena Äini ovakav tretman bezbednijim i efikasnijim. U ovom radu, dva od tri (anhidrid cis-akonitne, citrakonske i 2,3-dimetilmaleinske kiseline) ispitivana anhidrida su se pokazala pogodnim za hemijske modifikacije alergena. Art v 1 je modifikovan cis-akonitilovanjem i citrakonilovanjem u cilju dobijanja derivata Art v 1 pogodnih za dalje imunoloÅ”ke testove. Acilovanjem Art v 1 dobijeni su derivati (caaArt v 1 i citArt v 1) sa oko 80 % izmodifikovanih amino grupa. Dobijeni derivati su monomerni, sa molekulskom masom sliÄnom nativnom Art v 1, ali sa dramatiÄno smanjenim pI vrednostima. Oba derivata su relativno stabilna u neutralnoj, dok se u kiseloj sredini acil grupe hidrolizuju. Modifikacija alergena cis-akonitilovanjem i citrakonilovanjem može biti novi naÄin za dobijanje alergoida
The importance of cross-reactivity in grass pollen allergy
According to the data obtained from in vivo and in vitro testing in Serbia, a significant number of patients have allergic symptoms caused by grass pollen. We examined the protein composition of grass pollens (Dactylis glomerata, Lolium perenne and Phleum pratense) and cross-reactivity in patients allergic to grass pollen from our region. The grass pollen allergen extract was characterized by SDS-PAGE, while cross-reactivity of single grass pollens was revealed by immunoblot analysis. A high degree of cross-reactivity was demonstrated for all three single pollens in the sera of allergic patients compared to the grass pollen extract mixture. Confirmation of the existence of cross-reactivity between different antigenic sources facilitates the use of monovalent vaccines, which are easier to standardize and at the same time prevent further sensitization of patients and reduces adverse reactions
Supplementary data for article: GrozdanoviÄ, M. M.; Burazer, L. M.; GavroviÄ-JankuloviÄ, M. Kiwifruit (Actinidia Deliciosa) Extract Shows Potential as a Low-Cost and Efficient Milk-Clotting Agent. International Dairy Journal 2013, 32 (1), 46ā52. https://doi.org/10.1016/j.idairyj.2013.03.001
Supplementary material for: [https://doi.org/10.1016/j.idairyj.2013.03.001]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1589
Optimization of the heterologous expression of banana glucanase in Escherichia coli (vol 77, pg 43, 2012)
For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for production of the protein induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37 C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25 degrees C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of about 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.Za potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u Äelijama je indukovana 1 mM izopropil-Ī²-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37Ā°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja Äelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem Äelija nakon dodatka IPTG na 25Ā°C tokom 12 h. Rekombinantni GST-Mus a 5 preÄiÅ”Äen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrÄena je u 'dot blot' sa pojedinaÄnim serumima osoba alergiÄnih na bananu, i sa poliklonskim zeÄijim antitelima na ekstrakt banane, redom. PreÄiÅ”Äena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu
Optimizacija heterologe proizvodnje glukanaze iz banane u E. coli
For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pG EX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 con struct were employed for production of the protein induced by 1 mM isopropyl-Ī²-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37Ā°C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25Ā°C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of a bout 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot an analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.Za potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u Äelijama je indukovana 1 mM izopropil-Ī²-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37Ā°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja Äelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem Äelija nakon dodatka IPTG na 25Ā°C tokom 12 h. Rekombinantni GST-Mus a 5 preÄiÅ”Äen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrÄena je u 'dot blot' sa pojedinaÄnim serumima osoba alergiÄnih na bananu, i sa poliklonskim zeÄijim antitelima na ekstrakt banane, redom. PreÄiÅ”Äena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu
Evaluacija kriterijuma za dijagnozu atopijskog dermatitisa i detekcija alergen specifiÄnih IgE antitela kod pasa alergiÄnih 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 najÄeÅ”Äih uzroÄnika alergijskih reakcija izazvanih polenom kod ljudi i pasa. JoÅ” uvek nije definisano koji je glavni alergen (i), na koji, veÄina pasa alergiÄnih 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, ukljuÄujuÄi intradermalni test i dokazivanje prisustva alergen specifiÄnih IgE antitela u serumu. Optimizovani su uslovi za detekciju IgE specifiÄnih antitela iz seruma pasa alergiÄnih na polen ambrozije imunoblot tehnikom. Psi koji su imali pozitivnu reakciju na polen ove biljke na intradermalnom testu, takoÄe su imali specifiÄna 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 specifiÄnoj imunoterapiji obolelih pasa. TakoÄe je razmatrana i validnost Favrotovih dijagnostiÄkih kriterijuma kod pasa alergiÄnih na polen ambrozije. Može se zakljuÄiti da su predloženi kriterijumi od velike pomoÄi u postavljanju suspektne dijagnoze atopijskog dermatitisa pasa, nakon iskljuÄenja drugih pruritiÄnih dermatoza
Izolovanje i karakterizacija 68 kD alergena iz ekstrakta kuÄnih grinja
House dust mites (HDM) represent a major source of allergens, contributing to the increasing incidence of type I hypersensitivity disease worldwide. Over 30 different IgE-binding proteins from the HDM extract were detected. Although group 1 and 2 have been identified as major allergens, due to the safety and efficacy of allergy diagnosis and immunotherapy, there is a need to carefully evaluate the clinical relevance of other allergens present in the HDM extract. In regard to this, a high molecular mass allergen of about 68 kD was purified from the HDM extract using a combination of gel permeation chromatography and reversed-phase chromatography. The IgG and IgE reactivity of the purified protein were preserved during the purification process, as confirmed by Western blot analysis with polyclonal rabbit antibodies and dot blot analysis with a pool of sera from subjects with house dust mite allergy, respectively. In addition, the IgE reactivity was confirmed using ELISA testing with nine patient sera. The biological potency of the 68 kD allergen was confirmed by skin prick testing in five allergic subjects, suggesting that the high molecular mass allergen is a good candidate for component-resolved diagnosis of house dust mite allergy and eventual therapeutic treatment.Grinje iz kuÄne praÅ”ine predstavljaju jedan od glavnih izvora alergena koji su u znaÄajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujuÄih proteina iz kuÄne praÅ”ine je detektovano do danas. Alergeni grupe 1 i 2 oznaÄeni su kao glavni alergeni kuÄne praÅ”ine. MeÄutim, da bi se poboljÅ”ala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kuÄne praÅ”ine, neophodno je odrediti kliniÄki znaÄaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kuÄne praÅ”ine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost preÄiÅ”Äenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zeÄijim antitelima na ekstrakt kuÄne praÅ”ine i 'pool'-om seruma osoba alergiÄnih na kuÄnu praÅ”inu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na preÄiÅ”Äeni protein u ELISA testu. BioloÅ”ka reaktivnost preÄiÅ”Äenog alergena je potvrÄena u kožnim probama na pet pacijenata, ukazujuÄi da je preÄiÅ”Äen alergen dobar kandidat za dijagnozu alergije na kuÄnu praÅ”inu pojedinaÄnim komponentama i eventualni terapeutski tretman
Digestomics of Cow's Milk: Short Digestion-Resistant Peptides of Casein Form Functional Complexes by Aggregation
The aim of this study was to identify short digestion-resistant peptides (SDRPs) released by pepsin digestion of the whole cow's milk and examine their IgE reactivity and allergenicity. Raw milk was subjected to simulated gastric digestion. SDRPs were fractionated from the digests and identified by MS. Milk SDRPs were evaluated for aggregability, propensity to compete for IgE binding with individual milk allergens, and ability to bind IgG4 from allergic and milk-tolerant individuals. The majority of milk SDRPs originated from caseins (97% of peptides) and overlapped with the known IgE epitopes of cow's milk allergens. SDRPs competed with milk proteins for binding to human IgE and readily formed aggregates. The average peptide length was 10.6 +/- 3.5 amino acids. The ability to provoke allergenic in vivo responses was confirmed by skin-prick testing (SPT) in five milk-allergic subjects. This was attributed to the peptide ability to aggregate into non-covalent complexes. SDRPs are able to induce response in SPT, but only in 50% of the sera SDRPs were able to inhibit IgG4 binding to caseins. Hence, SDRPs corresponding to the mainly continuous epitopes of milk proteins induce allergenic in vivo responses in milk-allergic subjects due to aggregation
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