TY  - CONF
AU  - Ivanović, Saša
AU  - Ćupić, Vitomir
AU  - Živković, Irena
AU  - Milovanović, Vladimir
AU  - Ćupić Miladinović, Dejana
AU  - Borozan, Sunčica
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/845
AB  - Poskok (Vipera ammodytes) predstavlja najzastupljeniju zmiju otrovnicu u Srbiji i na Balkanu, kako po brojnosti tako i po arealu rasprostranjenosti. U poređenju sa venomima ostalih otrovnica iz familije Viperidae na Balkanu, toksičnost venoma poskoka je najveća. Neurotoksičnost sirovog venoma poskoka smo ispitivali u in vitro uslovima na osnovu kontrakcija preparata dijafragme izazvanih poljnom električnom stimulacijom i aktivnosti enzima: acetilholinesteraze (AChE) i ukupnih ATP-aza u dijafragmi.Formirano je 5 grupa pacova (n=5): kontrola (dijafragma bez venoma), dijafragma sa venomom, dijafragma sa smešom venom/antivenom („Viekvin“, Torlak, Srbija) u odnosima 1:2, 1:10 i 1:20 (m/m). Kontrakcije dijafragme su opale na 50% kontrolnih kontrakcija: pod uticajem venoma za 62,00±2,31minuta, a sa smešom venom/antivenom 1:2 za 78,50±7,51 minuta, 1:10 za 150,00±17,32 minuta, 1:20 za 307,50±2,89 minuta. Statistička razlika (p0,05) pod dejstvom venoma. Venom inhibira aktivnost ukupnih ATP-aza dijafragme za 51,81% u odnosu na kontrolu. Venom poskoka ne deluje na AChE i nikotinske receptore u neuro-mišićnoj sinapsi dijafragme, već narušava energetski metabolizam mitohondrija.
AB  - Vipera ammodytes is the most common venomous snake in Serbia and the Balkans, both in terms of numbers and area of distribution. Compared to the venoms of other poisonous snakes from the family Viperidae in the Balkans, the toxicity of the V. ammodytes venom is the highest. We investigated the neurotoxicity of crude venom in vitro using contractions of the diaphragm preparation induced by electric field stimulation and enzyme activity: acetylcholinesterase (AChE) and total ATPases in the diaphragm. Five groups of rats were formed (n=5): control (diaphragm without venom), diaphragmwith venom, diaphragm with mixture of venom/antivenom ("Viekvin", Torlak, Serbia) in the ratio 1:2, 1:10 and 1:20 (m/m). Diaphragmatic contractions decreased to 50% of control contractions: under the influence of venom for 62.00±2.31 minutes, and with venom/antivenom mixture 1:2 for 78.50±7.51 minutes, 1:10 for 150.00±17.32 minutes, 1:20 for 307.50±2.89 minutes. A statistical difference (p 0.05) under the influence of the venom. Venom inhibits the total ATPases activity of the diaphragm by 51.81% compared with control.The venom of V. ammodytes does not act on AChE and nicotinic receptors in the neuromuscular synapse of the diaphragm, but interferes with mitochondrial energy metabolism.
PB  - Belgrade : Serbian Society of Toxicology
C3  - 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023
T1  - Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova
T1  - Neurotoxic effect of Vipera ammodytes venom on the rat isolated diaphragm model
EP  - 108
SP  - 107
UR  - https://hdl.handle.net/21.15107/rcub_intor_845
ER  - 

@conference{
author = "Ivanović, Saša and Ćupić, Vitomir and Živković, Irena and Milovanović, Vladimir and Ćupić Miladinović, Dejana and Borozan, Sunčica",
year = "2023",
abstract = "Poskok (Vipera ammodytes) predstavlja najzastupljeniju zmiju otrovnicu u Srbiji i na Balkanu, kako po brojnosti tako i po arealu rasprostranjenosti. U poređenju sa venomima ostalih otrovnica iz familije Viperidae na Balkanu, toksičnost venoma poskoka je najveća. Neurotoksičnost sirovog venoma poskoka smo ispitivali u in vitro uslovima na osnovu kontrakcija preparata dijafragme izazvanih poljnom električnom stimulacijom i aktivnosti enzima: acetilholinesteraze (AChE) i ukupnih ATP-aza u dijafragmi.Formirano je 5 grupa pacova (n=5): kontrola (dijafragma bez venoma), dijafragma sa venomom, dijafragma sa smešom venom/antivenom („Viekvin“, Torlak, Srbija) u odnosima 1:2, 1:10 i 1:20 (m/m). Kontrakcije dijafragme su opale na 50% kontrolnih kontrakcija: pod uticajem venoma za 62,00±2,31minuta, a sa smešom venom/antivenom 1:2 za 78,50±7,51 minuta, 1:10 za 150,00±17,32 minuta, 1:20 za 307,50±2,89 minuta. Statistička razlika (p0,05) pod dejstvom venoma. Venom inhibira aktivnost ukupnih ATP-aza dijafragme za 51,81% u odnosu na kontrolu. Venom poskoka ne deluje na AChE i nikotinske receptore u neuro-mišićnoj sinapsi dijafragme, već narušava energetski metabolizam mitohondrija., Vipera ammodytes is the most common venomous snake in Serbia and the Balkans, both in terms of numbers and area of distribution. Compared to the venoms of other poisonous snakes from the family Viperidae in the Balkans, the toxicity of the V. ammodytes venom is the highest. We investigated the neurotoxicity of crude venom in vitro using contractions of the diaphragm preparation induced by electric field stimulation and enzyme activity: acetylcholinesterase (AChE) and total ATPases in the diaphragm. Five groups of rats were formed (n=5): control (diaphragm without venom), diaphragmwith venom, diaphragm with mixture of venom/antivenom ("Viekvin", Torlak, Serbia) in the ratio 1:2, 1:10 and 1:20 (m/m). Diaphragmatic contractions decreased to 50% of control contractions: under the influence of venom for 62.00±2.31 minutes, and with venom/antivenom mixture 1:2 for 78.50±7.51 minutes, 1:10 for 150.00±17.32 minutes, 1:20 for 307.50±2.89 minutes. A statistical difference (p 0.05) under the influence of the venom. Venom inhibits the total ATPases activity of the diaphragm by 51.81% compared with control.The venom of V. ammodytes does not act on AChE and nicotinic receptors in the neuromuscular synapse of the diaphragm, but interferes with mitochondrial energy metabolism.",
publisher = "Belgrade : Serbian Society of Toxicology",
journal = "13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023",
title = "Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova, Neurotoxic effect of Vipera ammodytes venom on the rat isolated diaphragm model",
pages = "108-107",
url = "https://hdl.handle.net/21.15107/rcub_intor_845"
}

Ivanović, S., Ćupić, V., Živković, I., Milovanović, V., Ćupić Miladinović, D.,& Borozan, S.. (2023). Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova. in 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023
Belgrade : Serbian Society of Toxicology., 107-108.
https://hdl.handle.net/21.15107/rcub_intor_845

Ivanović S, Ćupić V, Živković I, Milovanović V, Ćupić Miladinović D, Borozan S. Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova. in 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023. 2023;:107-108.
https://hdl.handle.net/21.15107/rcub_intor_845 .

Ivanović, Saša, Ćupić, Vitomir, Živković, Irena, Milovanović, Vladimir, Ćupić Miladinović, Dejana, Borozan, Sunčica, "Neurotoksično dejstvo venoma Vipera ammodytes na modelu izolovane dijafragme pacova" in 13th International Congress of the Serbian Society of Toxicology and 1st TOXSEE Regional Conference, belgrade, 10 - 12 May, 2023 (2023):107-108,
https://hdl.handle.net/21.15107/rcub_intor_845 .

TY  - JOUR
AU  - Milovanović, Vladimir
AU  - Minić, Rajna
AU  - Vakić, J.
AU  - Ivanović, S.
AU  - Cupić, V.
AU  - Borozan, S.
AU  - Nešić, A.
AU  - Živković, Irena
PY  - 2021
UR  - http://intor.torlakinstitut.com/handle/123456789/572
AB  - The MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms.
PB  - Elsevier Ltd
T2  - Toxicon
T1  - MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation
EP  - 65
SP  - 57
VL  - 192
DO  - 10.1016/j.toxicon.2021.01.012
ER  - 

@article{
author = "Milovanović, Vladimir and Minić, Rajna and Vakić, J. and Ivanović, S. and Cupić, V. and Borozan, S. and Nešić, A. and Živković, Irena",
year = "2021",
abstract = "The MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms.",
publisher = "Elsevier Ltd",
journal = "Toxicon",
title = "MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation",
pages = "65-57",
volume = "192",
doi = "10.1016/j.toxicon.2021.01.012"
}

Milovanović, V., Minić, R., Vakić, J., Ivanović, S., Cupić, V., Borozan, S., Nešić, A.,& Živković, I.. (2021). MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation. in Toxicon
Elsevier Ltd., 192, 57-65.
https://doi.org/10.1016/j.toxicon.2021.01.012

Milovanović V, Minić R, Vakić J, Ivanović S, Cupić V, Borozan S, Nešić A, Živković I. MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation. in Toxicon. 2021;192:57-65.
doi:10.1016/j.toxicon.2021.01.012 .

Milovanović, Vladimir, Minić, Rajna, Vakić, J., Ivanović, S., Cupić, V., Borozan, S., Nešić, A., Živković, Irena, "MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation" in Toxicon, 192 (2021):57-65,
https://doi.org/10.1016/j.toxicon.2021.01.012 . .

TY  - JOUR
AU  - Milovanović, Vladimir
AU  - Dimitrijević, Ljiljana
AU  - Petrušić, Vladimir
AU  - Kadrić, Jasminka
AU  - Minić, Rajna
AU  - Živković, Irena
PY  - 2018
UR  - http://intor.torlakinstitut.com/handle/123456789/500
AB  - During time, both professionals and general public became aware of the importance of animal welfare. This term not only covers endangered wild animal species, animals used in food industry, pets and experimental animals, but also animals used in production of biologics. The implementation of the 3R concept (Replacement, Reduction and Refinement) is especially important in this type of production. In this article, we describe for the first time the low dose, low volume and multi-site immunization protocol, as well as appropriate ELISA we developed for production of European anti-viper (V. ammodytes, long horned) antivenom in horses, which can help to significantly improve the welfare of the used animals.
PB  - Univerzitet u Beogradu - Fakultet veterinarske medicine, Beograd
T2  - Acta veterinaria - Beograd
T1  - Application of the 3r concept in the production of European antiviperinum on horses - multisite, low volumes immunization protocol and Elisa
EP  - 419
IS  - 4
SP  - 401
VL  - 68
DO  - 10.2478/acve-2018-0033
ER  - 

@article{
author = "Milovanović, Vladimir and Dimitrijević, Ljiljana and Petrušić, Vladimir and Kadrić, Jasminka and Minić, Rajna and Živković, Irena",
year = "2018",
abstract = "During time, both professionals and general public became aware of the importance of animal welfare. This term not only covers endangered wild animal species, animals used in food industry, pets and experimental animals, but also animals used in production of biologics. The implementation of the 3R concept (Replacement, Reduction and Refinement) is especially important in this type of production. In this article, we describe for the first time the low dose, low volume and multi-site immunization protocol, as well as appropriate ELISA we developed for production of European anti-viper (V. ammodytes, long horned) antivenom in horses, which can help to significantly improve the welfare of the used animals.",
publisher = "Univerzitet u Beogradu - Fakultet veterinarske medicine, Beograd",
journal = "Acta veterinaria - Beograd",
title = "Application of the 3r concept in the production of European antiviperinum on horses - multisite, low volumes immunization protocol and Elisa",
pages = "419-401",
number = "4",
volume = "68",
doi = "10.2478/acve-2018-0033"
}

Milovanović, V., Dimitrijević, L., Petrušić, V., Kadrić, J., Minić, R.,& Živković, I.. (2018). Application of the 3r concept in the production of European antiviperinum on horses - multisite, low volumes immunization protocol and Elisa. in Acta veterinaria - Beograd
Univerzitet u Beogradu - Fakultet veterinarske medicine, Beograd., 68(4), 401-419.
https://doi.org/10.2478/acve-2018-0033

Milovanović V, Dimitrijević L, Petrušić V, Kadrić J, Minić R, Živković I. Application of the 3r concept in the production of European antiviperinum on horses - multisite, low volumes immunization protocol and Elisa. in Acta veterinaria - Beograd. 2018;68(4):401-419.
doi:10.2478/acve-2018-0033 .

Milovanović, Vladimir, Dimitrijević, Ljiljana, Petrušić, Vladimir, Kadrić, Jasminka, Minić, Rajna, Živković, Irena, "Application of the 3r concept in the production of European antiviperinum on horses - multisite, low volumes immunization protocol and Elisa" in Acta veterinaria - Beograd, 68, no. 4 (2018):401-419,
https://doi.org/10.2478/acve-2018-0033 . .

TY  - CONF
AU  - Živković, Irena
AU  - Milovanović, Vladimir
AU  - Stojanović, Milan
AU  - Stanković, Aleksandra
AU  - Minić, Rajna
AU  - Dimitrijević, Ljiljana
PY  - 2017
UR  - http://intor.torlakinstitut.com/handle/123456789/713
AB  - Poznato je da 3R koncept u radu sa eksperimentalnim životinjama označava: smanjenje broja korišćenih životinja (Reduction), zamenu testova na
životinjama in vitro metodama (Replacement) i poboljšanje tretmana i
manipulacije (Refinement). Ovaj koncept je uspešno primenjen u postupku
produkcije zmijskog protivotrova (antivenoma) u Institutu za virusologiju,
vakcine i serume "Torlak".
Životinje izbora za imunizaciju u proizvodnji antivenoma su često konji
zbog mogućnosti produkcije velike (komercijalne) količine antivenoma što
se ne može lako postići na malim životinjama. Jedan od najvažnijih koraka u
produkciji antivenoma je imunizacija otrovom (venomom) na takav način
da se u organizmu životinje indukuje dugotrajan i visok titar specifičnih
antitela. Standardni imunizacioni protokol podrazumevao je injektovanje
velike količine emulzifikovanog otrova na jedno mesto, što je stvaralo
granulome i/ili rane na mestu ubrizgavanja. Novom šemom imunizacije,
aplikacijom male količine emulzije na mnogo mesta, ovakve neželjene
pojave su prestale ili postale zanemarljive (Refinement).
Nova šema imunizacije podrazumeva primenu doktrine "Mala doza, mala
količina-zapremina, mnogo mesta injektovanja" (" Low dose, low volume,
multi-site immunization "). Na taj način se smanjuje potreba za velikom
količinom otrova, zbog što posledično dovodi do smanjenja potrebe za
nabavkom velikog broja zmija, smanjenja rada sa zmijama, kao i smanjenja
opasnosti za radnika koji radi sa zmijama u više aspekata (kraći rad, manji
umor, smanjena šansa za eksces). Nova šema imunizacije je ujedno poboljšala titar antitela, a time povećala količinu antiseruma koja se može
produkovati u jednom konju, što dalje dovodi do smanjenja broja konja
potrebnih za imunizaciju (Reduction).
Paralelno sa razvijanjem nove imunizacione šeme, u laboratoriji je razvijen
ELISA test za detekciju IgG antitela specifičnih za venom. Indukcija i
povećanje specifičnih antitela, standardno se prati testom potence na
miševima, kojim se meri koliku zaštitu produkovan konjski antiserum pruža
miševima koji su primili letalnu dozu otrova. Pokazali smo da ELISA test može da zameni test potence tokom imunizacionog perioda i perioda
održavanja titra (Replacement).
Novi imunizacioni protokol i razvijen laboratorijski metod za praćenje
kinetike specifičnih antivenom antitela, omogućili su ne samo pojednostavljenje produkcionog postupka i povećanje prinosa proizvoda, već i
znatno poboljšanje dobrobiti eksperimentalnih životinja, naročito konja.
Ovako uspešan pristup konceptu 3R može se primeniti i u proizvodnji
drugih imunih seruma na konjima.
PB  - Univerzitet u Beogradu, Fakultet veterinarske medicine
C3  - Sedmo regionalno Savetovanje uzgoj, reprodukcija i zdravstvena zaštita konja, Zbornik predavanja, Ljubičevo
T1  - Primena koncepta 3r u produkciji antivenoma na konjima
EP  - 118
SP  - 117
UR  - https://hdl.handle.net/21.15107/rcub_intor_713
ER  - 

@conference{
author = "Živković, Irena and Milovanović, Vladimir and Stojanović, Milan and Stanković, Aleksandra and Minić, Rajna and Dimitrijević, Ljiljana",
year = "2017",
abstract = "Poznato je da 3R koncept u radu sa eksperimentalnim životinjama označava: smanjenje broja korišćenih životinja (Reduction), zamenu testova na
životinjama in vitro metodama (Replacement) i poboljšanje tretmana i
manipulacije (Refinement). Ovaj koncept je uspešno primenjen u postupku
produkcije zmijskog protivotrova (antivenoma) u Institutu za virusologiju,
vakcine i serume "Torlak".
Životinje izbora za imunizaciju u proizvodnji antivenoma su često konji
zbog mogućnosti produkcije velike (komercijalne) količine antivenoma što
se ne može lako postići na malim životinjama. Jedan od najvažnijih koraka u
produkciji antivenoma je imunizacija otrovom (venomom) na takav način
da se u organizmu životinje indukuje dugotrajan i visok titar specifičnih
antitela. Standardni imunizacioni protokol podrazumevao je injektovanje
velike količine emulzifikovanog otrova na jedno mesto, što je stvaralo
granulome i/ili rane na mestu ubrizgavanja. Novom šemom imunizacije,
aplikacijom male količine emulzije na mnogo mesta, ovakve neželjene
pojave su prestale ili postale zanemarljive (Refinement).
Nova šema imunizacije podrazumeva primenu doktrine "Mala doza, mala
količina-zapremina, mnogo mesta injektovanja" (" Low dose, low volume,
multi-site immunization "). Na taj način se smanjuje potreba za velikom
količinom otrova, zbog što posledično dovodi do smanjenja potrebe za
nabavkom velikog broja zmija, smanjenja rada sa zmijama, kao i smanjenja
opasnosti za radnika koji radi sa zmijama u više aspekata (kraći rad, manji
umor, smanjena šansa za eksces). Nova šema imunizacije je ujedno poboljšala titar antitela, a time povećala količinu antiseruma koja se može
produkovati u jednom konju, što dalje dovodi do smanjenja broja konja
potrebnih za imunizaciju (Reduction).
Paralelno sa razvijanjem nove imunizacione šeme, u laboratoriji je razvijen
ELISA test za detekciju IgG antitela specifičnih za venom. Indukcija i
povećanje specifičnih antitela, standardno se prati testom potence na
miševima, kojim se meri koliku zaštitu produkovan konjski antiserum pruža
miševima koji su primili letalnu dozu otrova. Pokazali smo da ELISA test može da zameni test potence tokom imunizacionog perioda i perioda
održavanja titra (Replacement).
Novi imunizacioni protokol i razvijen laboratorijski metod za praćenje
kinetike specifičnih antivenom antitela, omogućili su ne samo pojednostavljenje produkcionog postupka i povećanje prinosa proizvoda, već i
znatno poboljšanje dobrobiti eksperimentalnih životinja, naročito konja.
Ovako uspešan pristup konceptu 3R može se primeniti i u proizvodnji
drugih imunih seruma na konjima.",
publisher = "Univerzitet u Beogradu, Fakultet veterinarske medicine",
journal = "Sedmo regionalno Savetovanje uzgoj, reprodukcija i zdravstvena zaštita konja, Zbornik predavanja, Ljubičevo",
title = "Primena koncepta 3r u produkciji antivenoma na konjima",
pages = "118-117",
url = "https://hdl.handle.net/21.15107/rcub_intor_713"
}

Živković, I., Milovanović, V., Stojanović, M., Stanković, A., Minić, R.,& Dimitrijević, L.. (2017). Primena koncepta 3r u produkciji antivenoma na konjima. in Sedmo regionalno Savetovanje uzgoj, reprodukcija i zdravstvena zaštita konja, Zbornik predavanja, Ljubičevo
Univerzitet u Beogradu, Fakultet veterinarske medicine., 117-118.
https://hdl.handle.net/21.15107/rcub_intor_713

Živković I, Milovanović V, Stojanović M, Stanković A, Minić R, Dimitrijević L. Primena koncepta 3r u produkciji antivenoma na konjima. in Sedmo regionalno Savetovanje uzgoj, reprodukcija i zdravstvena zaštita konja, Zbornik predavanja, Ljubičevo. 2017;:117-118.
https://hdl.handle.net/21.15107/rcub_intor_713 .

Živković, Irena, Milovanović, Vladimir, Stojanović, Milan, Stanković, Aleksandra, Minić, Rajna, Dimitrijević, Ljiljana, "Primena koncepta 3r u produkciji antivenoma na konjima" in Sedmo regionalno Savetovanje uzgoj, reprodukcija i zdravstvena zaštita konja, Zbornik predavanja, Ljubičevo (2017):117-118,
https://hdl.handle.net/21.15107/rcub_intor_713 .