TY  - DATA
AU  - Mladenović Stokanić, Maja
AU  - Simović, Ana
AU  - Jovanović, Vesna
AU  - Radomirović, Mirjana
AU  - Udovički, Božidar
AU  - Krstić Ristivojević, Maja
AU  - Djukić, Teodora
AU  - Vasović, Tamara
AU  - Aćimović, Jelena
AU  - Sabljić, Ljiljana
AU  - Lukić, Ivana
AU  - Kovačević, Ana
AU  - Cujic, Danica
AU  - Gnjatović, Marija
AU  - Smiljanić, Katarina
AU  - Stojadinović, Marija
AU  - Radosavljević, Jelena
AU  - Stanić-Vučinić, Dragana
AU  - Stojanović, Marijana
AU  - Rajković, Andreja
AU  - Ćirkovic Veličković, Tanja
PY  - 2024
UR  - http://intor.torlakinstitut.com/handle/123456789/859
AB  - S1.1. Checking of N protein purity Recombinant N protein purity was checked after imidazole removal and buffer exchange by SDS PAGE (Figure 6.). For comparison, commercial high-purity HSA was also analyzed. S1.2. Identification of N protein Tandem mass spectrometry identification of proteins in an in-gel digested band of N protein (Figure S1, lane 3), confirmed the identity of N protein with high scores and peptide coverage (Fig. S2.). S2. Purification of polyclonal antibodies from mice and rabbit sera For the development of an ELISA test specific for the detection of SARS-CoV-2 N protein, recombinantly produced N protein was used for the immunization of mice and rabbits. Sera obtained from rabbits and mice were then tested for titer and specificity (Figure S3 and Figure 1). To determine the titer of polyclonal sera required to detect N protein in samples, we use wells coated with N protein and serial dilution of sera pools from different animals. After multiple washing steps, we detected the binding of rabbit and mice antibodies using secondary biotinylated antibodies and streptavidin-alkaline phosphatase chimaera or secondary antibodies with previously coupled alkaline phosphatase, where the amount of enzymes’ substrate converted to the product was measured as an increase in absorbance at 405 nm. As shown in Figure S3A, unpurified sera pools from both animals showed very high titers and expected logarithmic decrease of signal with dilution. Based on the obtained data titer for unpurified sera was determined to be X. The same trend was observed for pools purified using AS precipitation and rabbit sera purified using protein A affinity chromatography (Figure S3B and S3C). As shown in Figure S3D, clear bands from antibodies could be observed in both full and purified samples. Western blot analysis showed only one protein band on mass around 40 kDa, a Accession number / Protein Name Score Coverage (%) Unique peptides P0DTC9|NCAP_SARS2 Nucleoprotein OS=Severe acute respiratory syndrome coronavirus 2, 46 kDa 504.9 74.22 183 mass of purified N protein suggesting that the obtained sera is highly specific for N protein (Figure 2). Section S3 Diagnostic validationS3.1. Stabilization of capture antibodies Pre-coated ELISA plates were prepared for usage in clinical practice. To ensure the preservation of the biofunctionality of the surface-bound capture antibodies, the commonly used stabilizing excipient, 3% sucrose with 10% glycerol in MilliQ water was used. The plates were incubated with 300 μL per well of a stabilizing agent for 1 hour at room temperature. After an hour of incubation, the solution was carefully aspirated from each well. The plate was then blotted against clear paper towels to remove any remaining liquid, and the plates were allowed to air dry for 3 hours at RT. Dried plates were wrapped in parafilm and stored at 4 °C for later use. To remove the stabilizing agent coating, wells were washed with slightly acidic distilled water (pH of 6) three times, leaving the plate prepared for subsequent assay steps. Section S4. Characterization of N protein by HRMS S4.1. SDS PAGE and in-gel digestion Characterization of the produced recombinant N protein was done by HRMS after its in-gel digestion. A total of 10 μg of purified protein(s) were loaded in a 0.5 cm wide well and after SDSPAGE gel was stained with Coomassie Brilliant Blue R-250 (CBB). Protein gel bands were washed, reduced with dithiothreitol, and alkylated with iodoacetamide, followed by in-gel trypsin digestion1 (Shevchenko et al. 2006) with some minor modifications. The amount of trypsin was leveled to a trypsin/sample ratio of 1:30 (w/w). The final concentration of MS-grade trypsin (diluted in 25 mM ammonium bicarbonate buffer) was 1 ng/μL. Sample clean-up was performed using zip tips HyperSep C18 (Thermo Fisher Scientific Inc., Bremen, Germany). S5.1 Immunization of rabbits and mice Mice immunization Swiss Webster mice (n=10) were immunized subcutaneously with N protein formulated with Complete Freund`s adjuvant (CFA; 1st dose, 100 μg N protein / dose) or Incomplete Freund`s adjuvant (IFA; 2nd and 3rd doses, 50 μg N protein / dose) in three-week intervals. Mice were housed in small groups of up to six animals and had access to commercial mice food and water ad libitum. N protein solution (500ug/ml in PBS) was sterilized by filtering through 0.22 um filters. Sterile N protein solution was mixed with CFA (Sigma, Cat. No. F5881) at ratio 1:1 (v/v) under aseptic conditions. In total 400 ul of N protein-CFA emulsion (N protein final concentration 250ug/ml) was applied per immunization per mouse. Initial immunization was done by injection of N protein in CFA given subcutaneously (SC) in four sites (thigh pocket, base of tail, and mediastinum) with a 100 ul using 23-25 gauge needle. In total 100 ug of N protein was applied per mouse (25 ug per site). Subsequent immunizations with booster doses were done in the same way, but using IFA (Sigma, Cat. No. F5506) instead of CFA and N protein final concentration was 125 ug/ml. . In total 50 ug of N protein was applied per mouse (12.5 ug per site). Immunizations were done every three weeks. Mice immunization scheme: 1. day 0 – N protein in PBS: CFA = 1:1 (v/v); N protein final concentration was 250 μg/mL; 400 μL per mice (4x100 μL), e.g. 100 μg per mice 2. day 21 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μL per mice (4x100 μL), e.g. 50 μg per mice 3. day 42 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μl per mice (4x100 μL), e.g. 50 μg per mice First bleeding was performed two weeks after the 3rd dose, and then in intervals not shorter than two weeks. The sera obtained after the first bleeding was tested for the production of specific anti-N protein antibodies.
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - Supplementary information for the article:       Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333.
IS  - 1
VL  - 25
UR  - https://hdl.handle.net/21.15107/rcub_intor_859
ER  - 

@misc{
author = "Mladenović Stokanić, Maja and Simović, Ana and Jovanović, Vesna and Radomirović, Mirjana and Udovički, Božidar and Krstić Ristivojević, Maja and Djukić, Teodora and Vasović, Tamara and Aćimović, Jelena and Sabljić, Ljiljana and Lukić, Ivana and Kovačević, Ana and Cujic, Danica and Gnjatović, Marija and Smiljanić, Katarina and Stojadinović, Marija and Radosavljević, Jelena and Stanić-Vučinić, Dragana and Stojanović, Marijana and Rajković, Andreja and Ćirkovic Veličković, Tanja",
year = "2024",
abstract = "S1.1. Checking of N protein purity Recombinant N protein purity was checked after imidazole removal and buffer exchange by SDS PAGE (Figure 6.). For comparison, commercial high-purity HSA was also analyzed. S1.2. Identification of N protein Tandem mass spectrometry identification of proteins in an in-gel digested band of N protein (Figure S1, lane 3), confirmed the identity of N protein with high scores and peptide coverage (Fig. S2.). S2. Purification of polyclonal antibodies from mice and rabbit sera For the development of an ELISA test specific for the detection of SARS-CoV-2 N protein, recombinantly produced N protein was used for the immunization of mice and rabbits. Sera obtained from rabbits and mice were then tested for titer and specificity (Figure S3 and Figure 1). To determine the titer of polyclonal sera required to detect N protein in samples, we use wells coated with N protein and serial dilution of sera pools from different animals. After multiple washing steps, we detected the binding of rabbit and mice antibodies using secondary biotinylated antibodies and streptavidin-alkaline phosphatase chimaera or secondary antibodies with previously coupled alkaline phosphatase, where the amount of enzymes’ substrate converted to the product was measured as an increase in absorbance at 405 nm. As shown in Figure S3A, unpurified sera pools from both animals showed very high titers and expected logarithmic decrease of signal with dilution. Based on the obtained data titer for unpurified sera was determined to be X. The same trend was observed for pools purified using AS precipitation and rabbit sera purified using protein A affinity chromatography (Figure S3B and S3C). As shown in Figure S3D, clear bands from antibodies could be observed in both full and purified samples. Western blot analysis showed only one protein band on mass around 40 kDa, a Accession number / Protein Name Score Coverage (%) Unique peptides P0DTC9|NCAP_SARS2 Nucleoprotein OS=Severe acute respiratory syndrome coronavirus 2, 46 kDa 504.9 74.22 183 mass of purified N protein suggesting that the obtained sera is highly specific for N protein (Figure 2). Section S3 Diagnostic validationS3.1. Stabilization of capture antibodies Pre-coated ELISA plates were prepared for usage in clinical practice. To ensure the preservation of the biofunctionality of the surface-bound capture antibodies, the commonly used stabilizing excipient, 3% sucrose with 10% glycerol in MilliQ water was used. The plates were incubated with 300 μL per well of a stabilizing agent for 1 hour at room temperature. After an hour of incubation, the solution was carefully aspirated from each well. The plate was then blotted against clear paper towels to remove any remaining liquid, and the plates were allowed to air dry for 3 hours at RT. Dried plates were wrapped in parafilm and stored at 4 °C for later use. To remove the stabilizing agent coating, wells were washed with slightly acidic distilled water (pH of 6) three times, leaving the plate prepared for subsequent assay steps. Section S4. Characterization of N protein by HRMS S4.1. SDS PAGE and in-gel digestion Characterization of the produced recombinant N protein was done by HRMS after its in-gel digestion. A total of 10 μg of purified protein(s) were loaded in a 0.5 cm wide well and after SDSPAGE gel was stained with Coomassie Brilliant Blue R-250 (CBB). Protein gel bands were washed, reduced with dithiothreitol, and alkylated with iodoacetamide, followed by in-gel trypsin digestion1 (Shevchenko et al. 2006) with some minor modifications. The amount of trypsin was leveled to a trypsin/sample ratio of 1:30 (w/w). The final concentration of MS-grade trypsin (diluted in 25 mM ammonium bicarbonate buffer) was 1 ng/μL. Sample clean-up was performed using zip tips HyperSep C18 (Thermo Fisher Scientific Inc., Bremen, Germany). S5.1 Immunization of rabbits and mice Mice immunization Swiss Webster mice (n=10) were immunized subcutaneously with N protein formulated with Complete Freund`s adjuvant (CFA; 1st dose, 100 μg N protein / dose) or Incomplete Freund`s adjuvant (IFA; 2nd and 3rd doses, 50 μg N protein / dose) in three-week intervals. Mice were housed in small groups of up to six animals and had access to commercial mice food and water ad libitum. N protein solution (500ug/ml in PBS) was sterilized by filtering through 0.22 um filters. Sterile N protein solution was mixed with CFA (Sigma, Cat. No. F5881) at ratio 1:1 (v/v) under aseptic conditions. In total 400 ul of N protein-CFA emulsion (N protein final concentration 250ug/ml) was applied per immunization per mouse. Initial immunization was done by injection of N protein in CFA given subcutaneously (SC) in four sites (thigh pocket, base of tail, and mediastinum) with a 100 ul using 23-25 gauge needle. In total 100 ug of N protein was applied per mouse (25 ug per site). Subsequent immunizations with booster doses were done in the same way, but using IFA (Sigma, Cat. No. F5506) instead of CFA and N protein final concentration was 125 ug/ml. . In total 50 ug of N protein was applied per mouse (12.5 ug per site). Immunizations were done every three weeks. Mice immunization scheme: 1. day 0 – N protein in PBS: CFA = 1:1 (v/v); N protein final concentration was 250 μg/mL; 400 μL per mice (4x100 μL), e.g. 100 μg per mice 2. day 21 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μL per mice (4x100 μL), e.g. 50 μg per mice 3. day 42 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μl per mice (4x100 μL), e.g. 50 μg per mice First bleeding was performed two weeks after the 3rd dose, and then in intervals not shorter than two weeks. The sera obtained after the first bleeding was tested for the production of specific anti-N protein antibodies.",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "Supplementary information for the article:       Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333.",
number = "1",
volume = "25",
url = "https://hdl.handle.net/21.15107/rcub_intor_859"
}

Mladenović Stokanić, M., Simović, A., Jovanović, V., Radomirović, M., Udovički, B., Krstić Ristivojević, M., Djukić, T., Vasović, T., Aćimović, J., Sabljić, L., Lukić, I., Kovačević, A., Cujic, D., Gnjatović, M., Smiljanić, K., Stojadinović, M., Radosavljević, J., Stanić-Vučinić, D., Stojanović, M., Rajković, A.,& Ćirkovic Veličković, T.. (2024). Supplementary information for the article:       Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333.. in International Journal of Molecular Sciences
MDPI., 25(1).
https://hdl.handle.net/21.15107/rcub_intor_859

Mladenović Stokanić M, Simović A, Jovanović V, Radomirović M, Udovički B, Krstić Ristivojević M, Djukić T, Vasović T, Aćimović J, Sabljić L, Lukić I, Kovačević A, Cujic D, Gnjatović M, Smiljanić K, Stojadinović M, Radosavljević J, Stanić-Vučinić D, Stojanović M, Rajković A, Ćirkovic Veličković T. Supplementary information for the article:       Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333.. in International Journal of Molecular Sciences. 2024;25(1).
https://hdl.handle.net/21.15107/rcub_intor_859 .

Mladenović Stokanić, Maja, Simović, Ana, Jovanović, Vesna, Radomirović, Mirjana, Udovički, Božidar, Krstić Ristivojević, Maja, Djukić, Teodora, Vasović, Tamara, Aćimović, Jelena, Sabljić, Ljiljana, Lukić, Ivana, Kovačević, Ana, Cujic, Danica, Gnjatović, Marija, Smiljanić, Katarina, Stojadinović, Marija, Radosavljević, Jelena, Stanić-Vučinić, Dragana, Stojanović, Marijana, Rajković, Andreja, Ćirkovic Veličković, Tanja, "Supplementary information for the article:       Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333." in International Journal of Molecular Sciences, 25, no. 1 (2024),
https://hdl.handle.net/21.15107/rcub_intor_859 .

TY  - JOUR
AU  - Mladenović Stokanić, Maja
AU  - Simović, Ana
AU  - Jovanović, Vesna
AU  - Radomirović, Mirjana
AU  - Udovički, Božidar
AU  - Krstić Ristivojević, Maja
AU  - Djukić, Teodora
AU  - Vasović, Tamara
AU  - Aćimović, Jelena
AU  - Sabljić, Ljiljana
AU  - Lukić, Ivana
AU  - Kovačević, Ana
AU  - Cujic, Danica
AU  - Gnjatović, Marija
AU  - Smiljanić, Katarina
AU  - Stojadinović, Marija
AU  - Radosavljević, Jelena
AU  - Stanić-Vučinić, Dragana
AU  - Stojanović, Marijana
AU  - Rajković, Andreja
AU  - Ćirkovic Veličković, Tanja
PY  - 2024
UR  - http://intor.torlakinstitut.com/handle/123456789/858
AB  - In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species
IS  - 1
SP  - 333
VL  - 25
DO  - 10.3390/ijms25010333
ER  - 

@article{
author = "Mladenović Stokanić, Maja and Simović, Ana and Jovanović, Vesna and Radomirović, Mirjana and Udovički, Božidar and Krstić Ristivojević, Maja and Djukić, Teodora and Vasović, Tamara and Aćimović, Jelena and Sabljić, Ljiljana and Lukić, Ivana and Kovačević, Ana and Cujic, Danica and Gnjatović, Marija and Smiljanić, Katarina and Stojadinović, Marija and Radosavljević, Jelena and Stanić-Vučinić, Dragana and Stojanović, Marijana and Rajković, Andreja and Ćirkovic Veličković, Tanja",
year = "2024",
abstract = "In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species",
number = "1",
pages = "333",
volume = "25",
doi = "10.3390/ijms25010333"
}

Mladenović Stokanić, M., Simović, A., Jovanović, V., Radomirović, M., Udovički, B., Krstić Ristivojević, M., Djukić, T., Vasović, T., Aćimović, J., Sabljić, L., Lukić, I., Kovačević, A., Cujic, D., Gnjatović, M., Smiljanić, K., Stojadinović, M., Radosavljević, J., Stanić-Vučinić, D., Stojanović, M., Rajković, A.,& Ćirkovic Veličković, T.. (2024). Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. in International Journal of Molecular Sciences
MDPI., 25(1), 333.
https://doi.org/10.3390/ijms25010333

Mladenović Stokanić M, Simović A, Jovanović V, Radomirović M, Udovički B, Krstić Ristivojević M, Djukić T, Vasović T, Aćimović J, Sabljić L, Lukić I, Kovačević A, Cujic D, Gnjatović M, Smiljanić K, Stojadinović M, Radosavljević J, Stanić-Vučinić D, Stojanović M, Rajković A, Ćirkovic Veličković T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. in International Journal of Molecular Sciences. 2024;25(1):333.
doi:10.3390/ijms25010333 .

Mladenović Stokanić, Maja, Simović, Ana, Jovanović, Vesna, Radomirović, Mirjana, Udovički, Božidar, Krstić Ristivojević, Maja, Djukić, Teodora, Vasović, Tamara, Aćimović, Jelena, Sabljić, Ljiljana, Lukić, Ivana, Kovačević, Ana, Cujic, Danica, Gnjatović, Marija, Smiljanić, Katarina, Stojadinović, Marija, Radosavljević, Jelena, Stanić-Vučinić, Dragana, Stojanović, Marijana, Rajković, Andreja, Ćirkovic Veličković, Tanja, "Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species" in International Journal of Molecular Sciences, 25, no. 1 (2024):333,
https://doi.org/10.3390/ijms25010333 . .

TY  - JOUR
AU  - Trifunović, Sara
AU  - Smiljanić, Katarina
AU  - Sickmann, Albert
AU  - Solari, Fiorella Andrea
AU  - Kolarević, Stoimir
AU  - Divac Rankov, Aleksandra
AU  - Ljujic, Mila
PY  - 2022
UR  - http://intor.torlakinstitut.com/handle/123456789/781
AB  - Background: Although still considered a safer alternative to classical cigarettes, growing body of work points toharmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect ofe-cigarettes needs to be investigated in more detail considering their widespread use.Methods: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, withand without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cel-lular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performedvia high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry.Results: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however itnegatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletionin total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteinswere upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of post-translational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data areavailable via ProteomeXchange with identifier PXD032071.Conclusions: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMsalterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.
PB  - BMC Springer Nature
T2  - Respiratory Research
T1  - Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells
IS  - 191
VL  - 23
DO  - 10.1186/s12931-022-02102-w
ER  - 

@article{
author = "Trifunović, Sara and Smiljanić, Katarina and Sickmann, Albert and Solari, Fiorella Andrea and Kolarević, Stoimir and Divac Rankov, Aleksandra and Ljujic, Mila",
year = "2022",
abstract = "Background: Although still considered a safer alternative to classical cigarettes, growing body of work points toharmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect ofe-cigarettes needs to be investigated in more detail considering their widespread use.Methods: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, withand without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cel-lular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performedvia high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry.Results: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however itnegatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletionin total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteinswere upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of post-translational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data areavailable via ProteomeXchange with identifier PXD032071.Conclusions: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMsalterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.",
publisher = "BMC Springer Nature",
journal = "Respiratory Research",
title = "Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells",
number = "191",
volume = "23",
doi = "10.1186/s12931-022-02102-w"
}

Trifunović, S., Smiljanić, K., Sickmann, A., Solari, F. A., Kolarević, S., Divac Rankov, A.,& Ljujic, M.. (2022). Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells. in Respiratory Research
BMC Springer Nature., 23(191).
https://doi.org/10.1186/s12931-022-02102-w

Trifunović S, Smiljanić K, Sickmann A, Solari FA, Kolarević S, Divac Rankov A, Ljujic M. Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells. in Respiratory Research. 2022;23(191).
doi:10.1186/s12931-022-02102-w .

Trifunović, Sara, Smiljanić, Katarina, Sickmann, Albert, Solari, Fiorella Andrea, Kolarević, Stoimir, Divac Rankov, Aleksandra, Ljujic, Mila, "Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells" in Respiratory Research, 23, no. 191 (2022),
https://doi.org/10.1186/s12931-022-02102-w . .

TY  - CONF
AU  - Ljujic, Mila
AU  - Trifunović, Sara
AU  - Smiljanić, Katarina
AU  - Solari, Fiorella Andrea
AU  - Sickmann, Albert
AU  - Divac Rankov, Aleksandra
PY  - 2022
UR  - http://intor.torlakinstitut.com/handle/123456789/782
AB  - The COVID-19 pandemic caused by the SARS-CoV2 virus poses a global health threat with over 5 million deaths recorded. There is little understanding regarding SARS-CoV2 pathogenesis in the human airways and disease severity increases with age. Neutrophils are white blood cells found in large numbers in the airways of the lungs in severe COVID-19 patients. It is not known whether this influx of neutrophils into the airway has a protective or detrimental effect. We aim to understand the role of neutrophils during COVID-19 pathology, using an experimental infection model of the airway epithelium from the eldelry and children. To do this, we collect nasal airway cells from healthy elderly and children and grow them at air-liquid interface. Once differentiation and ciliation of these cells is reached, we infect the cells with SARS-CoV2 virus and allow neutrophils to migrate from the basolateral (blood) to the apical (air) side of the epithelium, similar to a physiological airway. Using flow cytometric analyses, we measure the expression of activation markers and the number of neutrophils that migrate across the epithelium of different ages in response to SARS-CoV2 infection. Preliminary work shows less viable neutrophils recovered from the elderly epithelium, more activated neutrophils when migrating through the elderly epithelium, as well as increased numbers of neutrophils remaining on the basolateral (blood) side of the elderly epithelium. These findings point to an inflammatory neutrophil phenotype influenced by the damaged elderly epithelium and supports the hypothesis that neutrophils are responsible for the severity of disease.
PB  - European Respiraotory Society (ERS)
C3  - European Respiratory Journal
T1  - Electronic cigarette liquids impair protein synthesis and alter proteomic profiles in V79 cells
IS  - 66
SP  - 506
VL  - 60
DO  - 10.1183/13993003.congress-2022.506
ER  - 

@conference{
author = "Ljujic, Mila and Trifunović, Sara and Smiljanić, Katarina and Solari, Fiorella Andrea and Sickmann, Albert and Divac Rankov, Aleksandra",
year = "2022",
abstract = "The COVID-19 pandemic caused by the SARS-CoV2 virus poses a global health threat with over 5 million deaths recorded. There is little understanding regarding SARS-CoV2 pathogenesis in the human airways and disease severity increases with age. Neutrophils are white blood cells found in large numbers in the airways of the lungs in severe COVID-19 patients. It is not known whether this influx of neutrophils into the airway has a protective or detrimental effect. We aim to understand the role of neutrophils during COVID-19 pathology, using an experimental infection model of the airway epithelium from the eldelry and children. To do this, we collect nasal airway cells from healthy elderly and children and grow them at air-liquid interface. Once differentiation and ciliation of these cells is reached, we infect the cells with SARS-CoV2 virus and allow neutrophils to migrate from the basolateral (blood) to the apical (air) side of the epithelium, similar to a physiological airway. Using flow cytometric analyses, we measure the expression of activation markers and the number of neutrophils that migrate across the epithelium of different ages in response to SARS-CoV2 infection. Preliminary work shows less viable neutrophils recovered from the elderly epithelium, more activated neutrophils when migrating through the elderly epithelium, as well as increased numbers of neutrophils remaining on the basolateral (blood) side of the elderly epithelium. These findings point to an inflammatory neutrophil phenotype influenced by the damaged elderly epithelium and supports the hypothesis that neutrophils are responsible for the severity of disease.",
publisher = "European Respiraotory Society (ERS)",
journal = "European Respiratory Journal",
title = "Electronic cigarette liquids impair protein synthesis and alter proteomic profiles in V79 cells",
number = "66",
pages = "506",
volume = "60",
doi = "10.1183/13993003.congress-2022.506"
}

Ljujic, M., Trifunović, S., Smiljanić, K., Solari, F. A., Sickmann, A.,& Divac Rankov, A.. (2022). Electronic cigarette liquids impair protein synthesis and alter proteomic profiles in V79 cells. in European Respiratory Journal
European Respiraotory Society (ERS)., 60(66), 506.
https://doi.org/10.1183/13993003.congress-2022.506

Ljujic M, Trifunović S, Smiljanić K, Solari FA, Sickmann A, Divac Rankov A. Electronic cigarette liquids impair protein synthesis and alter proteomic profiles in V79 cells. in European Respiratory Journal. 2022;60(66):506.
doi:10.1183/13993003.congress-2022.506 .

Ljujic, Mila, Trifunović, Sara, Smiljanić, Katarina, Solari, Fiorella Andrea, Sickmann, Albert, Divac Rankov, Aleksandra, "Electronic cigarette liquids impair protein synthesis and alter proteomic profiles in V79 cells" in European Respiratory Journal, 60, no. 66 (2022):506,
https://doi.org/10.1183/13993003.congress-2022.506 . .

TY  - JOUR
AU  - Stojić-Vukanić, Zorica
AU  - Pilipović, Ivan
AU  - Arsenović-Ranin, Nevena
AU  - Dimitrijević, Mirjana
AU  - Leposavić, Gordana
PY  - 2021
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/3946
UR  - http://intor.torlakinstitut.com/handle/123456789/624
AB  - The incidence of multiple sclerosis (MS) and susceptibility of animals to experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, decrease with aging. Generally, autoimmune diseases develop as the ultimate outcome of an imbalance between damaging immune responses against self and regulatory immune responses (keeping the former under control). Thus, in this review the age-related changes possibly underlying this balance were discussed. Specifically, considering the central role of T cells in MS/EAE, the impact of aging on overall functional capacity (reflecting both overall count and individual functional cell properties) of self-reactive conventional T cells (Tcons) and FoxP3+ regulatory T cells (Tregs), as the most potent immunoregulatory/suppressive cells, was analyzed, as well. The analysis encompasses three distinct compartments: thymus (the primary lymphoid organ responsible for the elimination of self-reactive T cells – negative selection and the generation of Tregs, compensating for imperfections of the negative selection), peripheral blood/lymphoid tissues (“afferent” compartment), and brain/spinal cord tissues (“target” compartment). Given that the incidence of MS and susceptibility of animals to EAE are greater in women/females than in age-matched men/males, sex as independent variable was also considered. In conclusion, with aging, sex-specific alterations in the balance of self-reactive Tcons/Tregs are likely to occur not only in the thymus/”afferent” compartment, but also in the “target” compartment, reflecting multifaceted changes in both T-cell types. Their in depth understanding is important not only for envisaging effects of aging, but also for designing interventions to slow-down aging without any adverse effect on incidence of autoimmune diseases.
PB  - Elsevier B.V.
T2  - Immunology Letters
T1  - Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases
EP  - 59
SP  - 42
VL  - 239
DO  - 10.1016/j.imlet.2021.08.003
ER  - 

@article{
author = "Stojić-Vukanić, Zorica and Pilipović, Ivan and Arsenović-Ranin, Nevena and Dimitrijević, Mirjana and Leposavić, Gordana",
year = "2021",
abstract = "The incidence of multiple sclerosis (MS) and susceptibility of animals to experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, decrease with aging. Generally, autoimmune diseases develop as the ultimate outcome of an imbalance between damaging immune responses against self and regulatory immune responses (keeping the former under control). Thus, in this review the age-related changes possibly underlying this balance were discussed. Specifically, considering the central role of T cells in MS/EAE, the impact of aging on overall functional capacity (reflecting both overall count and individual functional cell properties) of self-reactive conventional T cells (Tcons) and FoxP3+ regulatory T cells (Tregs), as the most potent immunoregulatory/suppressive cells, was analyzed, as well. The analysis encompasses three distinct compartments: thymus (the primary lymphoid organ responsible for the elimination of self-reactive T cells – negative selection and the generation of Tregs, compensating for imperfections of the negative selection), peripheral blood/lymphoid tissues (“afferent” compartment), and brain/spinal cord tissues (“target” compartment). Given that the incidence of MS and susceptibility of animals to EAE are greater in women/females than in age-matched men/males, sex as independent variable was also considered. In conclusion, with aging, sex-specific alterations in the balance of self-reactive Tcons/Tregs are likely to occur not only in the thymus/”afferent” compartment, but also in the “target” compartment, reflecting multifaceted changes in both T-cell types. Their in depth understanding is important not only for envisaging effects of aging, but also for designing interventions to slow-down aging without any adverse effect on incidence of autoimmune diseases.",
publisher = "Elsevier B.V.",
journal = "Immunology Letters",
title = "Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases",
pages = "59-42",
volume = "239",
doi = "10.1016/j.imlet.2021.08.003"
}

Stojić-Vukanić, Z., Pilipović, I., Arsenović-Ranin, N., Dimitrijević, M.,& Leposavić, G.. (2021). Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases. in Immunology Letters
Elsevier B.V.., 239, 42-59.
https://doi.org/10.1016/j.imlet.2021.08.003

Stojić-Vukanić Z, Pilipović I, Arsenović-Ranin N, Dimitrijević M, Leposavić G. Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases. in Immunology Letters. 2021;239:42-59.
doi:10.1016/j.imlet.2021.08.003 .

Stojić-Vukanić, Zorica, Pilipović, Ivan, Arsenović-Ranin, Nevena, Dimitrijević, Mirjana, Leposavić, Gordana, "Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases" in Immunology Letters, 239 (2021):42-59,
https://doi.org/10.1016/j.imlet.2021.08.003 . .

TY  - DATA
AU  - Dimitrijević, Mirjana
AU  - Arsenović-Ranin, Nevena
AU  - Bufan, Biljana
AU  - Nacka-Aleksić, Mirjana
AU  - Kosec, Duško
AU  - Pilipović, Ivan
AU  - Kotur-Stevuljević, Jelena
AU  - Simić, Ljubica
AU  - Sopta, Jelena
AU  - Leposavić, Gordana
PY  - 2020
UR  - http://intor.torlakinstitut.com/handle/123456789/647
AB  - Supplementary Fig. 1 Sex differences in the clinical and histological presentation of CIA. (a) A line graph indicates daily arthritic score  (mean ± SEM) from the 12th to the 39th day post-immunization (d.p.i.) in male (n = 9) and female (n = 10) CIA rats.  Mann–Whitney U test: * p ≤ 0.05, from the 17th to the 39th d.p.i. (b) Line graph indicates daily arthritic score  (mean ± SEM) from the 13th to the 21st day post-immunization (d.p.i.) in male and female CIA rats. Mann–Whitney U test: n = 8 rats/sex. * p ≤ 0.05.  Photographs show representative arthritic joints (arrows) of hind paws from male and female CIA rats.  (c) Photomicrographs of HE-stained sections of paraffin-embedded joints from male and female CIA rats show replacement  of the normal bone marrow cell populations by inflammatory cells. In females, numerous multinuclear giant cells (red arrows)  are present as opposed to male CIA rats. Original magnification × 400. The bar indicates 100 μm (PNG 2723 kb) Supplementary Fig. 2 Fluorescence minus one controls for flow cytometry analysis of CD11b/CCR2/CX3CR1 staining of splenocytes.  For setting cutoff boundaries, gates were controlled using fluorescence minus one (FMO) controls obtained by omitting a single  antibody from the labeling antibody cocktail. Flow cytometry dot plots represent FMO controls without anti-CX3CR1 or anti-CCR2  Abs within CD11b+ splenocytes (gated as shown in Fig. 3) isolated from CIA rats on the 21st day post-immunization (PNG 98 kb). Supplementary Fig. 3 Fluorescence minus one controls for flow cytometry analysis of CD11b/CD43/CCR2/CX3CR1 staining of peripheral blood cells.  For setting cutoff boundaries, gates were controlled using fluorescence minus one (FMO) controls obtained by omitting a single  antibody from the labeling antibody cocktail. Flow cytometry dot plots represent FMO controls without (upper) CD43 mAb within  CD11b+ peripheral blood cells (gated as shown in Fig. 4a) and (lower) anti-CX3CR1 or anti-CCR2 Abs within CD11b+CD43+  peripheral blood cells isolated from CIA rats on the 21st day post-immunization (PNG 157 kb). Supplementary Fig. 4 Sex differences in the activation of Th cells, Th17 cell function, and frequency of CD40+CD11b+ antigen presenting cells in  draining lymph nodes from CIA rats, popliteal draining lymph nodes (DLNs) were retrieved from male and female CIA rats on the  21st day post-immunization. (a) Scatter plots with bar indicate the frequencies of activated Th cells (CD25+Foxp3-CD4+) and  Th17 cells (IL-17+CD4+TCRαβ+) in DLNs from CIA rats and the concentration of IL-17 in supernatants of collagen type  II-stimulated and unstimulated (medium) DLN cell cultures from male and female rats (see MATERIAL AND METHODS).  Linear graph shows the correlation between the frequency of activated Th cells (CD25+Foxp3-CD4+) and the frequency of  Th17 cells (IL-17+CD4+TCRαβ+) in DLNs from CIA rats. Pearson’s r value is shown in the graph. (b) Representative flow  cytometry dot plots show (upper) CD11b staining and (lower) CD40 vs CD11b staining of DLN cells from male and female rats.  Number indicates percent in the region. Scatter plots with bar indicate the frequency and the number of (upper) CD11b+ cells  and (lower) CD40+CD11b+ cells in DLNs of male and female rats. The Number indicates percent in the region. Results are  expressed as mean ± SEM. (c) The linear graph shows the correlation between the frequency of activated Th cells  (CD25+Foxp3-CD4+) and the frequency of CD40+CD11b+ cells in DLNs from CIA rats. Pearson’s r value is shown in the graph.  n = 8 rats/sex. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 (PNG 590 kb). Supplementary Fig. 5 Gating strategy for activated Th cells and Th17 cells, popliteal draining lymph nodes (DLNs) were retrieved from CIA rats on  the 21st day post-immunization. Flow cytometry dot plots show gating strategy for (a) activated The cells (CD25+Foxp3-CD4+)  and (b) Th17 cells (IL-17+CD4+TCRαβ+) (PNG 101 kb).
PB  - Springer
T2  - Inflammation
T1  - Supplementary information for the article: Dimitrijević, M.; Arsenović-Ranin, N.; Bufan, B.; Nacka-Aleksić, M.; Kosec, D.;  Pilipović, I.; Kotur-Stevuljević, J.; Simić, L.; Sopta, J.; Leposavić, G. Sex-Based Differences in Monocytic Lineage Cells  Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. Inflammation 2020, 43 (6),  2312–2331. https://doi.org/10.1007/s10753-020-01302-0.
EP  - 2331
IS  - 6
SP  - 2312
VL  - 43
UR  - https://hdl.handle.net/21.15107/rcub_intor_647
ER  - 

@misc{
author = "Dimitrijević, Mirjana and Arsenović-Ranin, Nevena and Bufan, Biljana and Nacka-Aleksić, Mirjana and Kosec, Duško and Pilipović, Ivan and Kotur-Stevuljević, Jelena and Simić, Ljubica and Sopta, Jelena and Leposavić, Gordana",
year = "2020",
abstract = "Supplementary Fig. 1 Sex differences in the clinical and histological presentation of CIA. (a) A line graph indicates daily arthritic score  (mean ± SEM) from the 12th to the 39th day post-immunization (d.p.i.) in male (n = 9) and female (n = 10) CIA rats.  Mann–Whitney U test: * p ≤ 0.05, from the 17th to the 39th d.p.i. (b) Line graph indicates daily arthritic score  (mean ± SEM) from the 13th to the 21st day post-immunization (d.p.i.) in male and female CIA rats. Mann–Whitney U test: n = 8 rats/sex. * p ≤ 0.05.  Photographs show representative arthritic joints (arrows) of hind paws from male and female CIA rats.  (c) Photomicrographs of HE-stained sections of paraffin-embedded joints from male and female CIA rats show replacement  of the normal bone marrow cell populations by inflammatory cells. In females, numerous multinuclear giant cells (red arrows)  are present as opposed to male CIA rats. Original magnification × 400. The bar indicates 100 μm (PNG 2723 kb) Supplementary Fig. 2 Fluorescence minus one controls for flow cytometry analysis of CD11b/CCR2/CX3CR1 staining of splenocytes.  For setting cutoff boundaries, gates were controlled using fluorescence minus one (FMO) controls obtained by omitting a single  antibody from the labeling antibody cocktail. Flow cytometry dot plots represent FMO controls without anti-CX3CR1 or anti-CCR2  Abs within CD11b+ splenocytes (gated as shown in Fig. 3) isolated from CIA rats on the 21st day post-immunization (PNG 98 kb). Supplementary Fig. 3 Fluorescence minus one controls for flow cytometry analysis of CD11b/CD43/CCR2/CX3CR1 staining of peripheral blood cells.  For setting cutoff boundaries, gates were controlled using fluorescence minus one (FMO) controls obtained by omitting a single  antibody from the labeling antibody cocktail. Flow cytometry dot plots represent FMO controls without (upper) CD43 mAb within  CD11b+ peripheral blood cells (gated as shown in Fig. 4a) and (lower) anti-CX3CR1 or anti-CCR2 Abs within CD11b+CD43+  peripheral blood cells isolated from CIA rats on the 21st day post-immunization (PNG 157 kb). Supplementary Fig. 4 Sex differences in the activation of Th cells, Th17 cell function, and frequency of CD40+CD11b+ antigen presenting cells in  draining lymph nodes from CIA rats, popliteal draining lymph nodes (DLNs) were retrieved from male and female CIA rats on the  21st day post-immunization. (a) Scatter plots with bar indicate the frequencies of activated Th cells (CD25+Foxp3-CD4+) and  Th17 cells (IL-17+CD4+TCRαβ+) in DLNs from CIA rats and the concentration of IL-17 in supernatants of collagen type  II-stimulated and unstimulated (medium) DLN cell cultures from male and female rats (see MATERIAL AND METHODS).  Linear graph shows the correlation between the frequency of activated Th cells (CD25+Foxp3-CD4+) and the frequency of  Th17 cells (IL-17+CD4+TCRαβ+) in DLNs from CIA rats. Pearson’s r value is shown in the graph. (b) Representative flow  cytometry dot plots show (upper) CD11b staining and (lower) CD40 vs CD11b staining of DLN cells from male and female rats.  Number indicates percent in the region. Scatter plots with bar indicate the frequency and the number of (upper) CD11b+ cells  and (lower) CD40+CD11b+ cells in DLNs of male and female rats. The Number indicates percent in the region. Results are  expressed as mean ± SEM. (c) The linear graph shows the correlation between the frequency of activated Th cells  (CD25+Foxp3-CD4+) and the frequency of CD40+CD11b+ cells in DLNs from CIA rats. Pearson’s r value is shown in the graph.  n = 8 rats/sex. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 (PNG 590 kb). Supplementary Fig. 5 Gating strategy for activated Th cells and Th17 cells, popliteal draining lymph nodes (DLNs) were retrieved from CIA rats on  the 21st day post-immunization. Flow cytometry dot plots show gating strategy for (a) activated The cells (CD25+Foxp3-CD4+)  and (b) Th17 cells (IL-17+CD4+TCRαβ+) (PNG 101 kb).",
publisher = "Springer",
journal = "Inflammation",
title = "Supplementary information for the article: Dimitrijević, M.; Arsenović-Ranin, N.; Bufan, B.; Nacka-Aleksić, M.; Kosec, D.;  Pilipović, I.; Kotur-Stevuljević, J.; Simić, L.; Sopta, J.; Leposavić, G. Sex-Based Differences in Monocytic Lineage Cells  Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. Inflammation 2020, 43 (6),  2312–2331. https://doi.org/10.1007/s10753-020-01302-0.",
pages = "2331-2312",
number = "6",
volume = "43",
url = "https://hdl.handle.net/21.15107/rcub_intor_647"
}

Dimitrijević, M., Arsenović-Ranin, N., Bufan, B., Nacka-Aleksić, M., Kosec, D., Pilipović, I., Kotur-Stevuljević, J., Simić, L., Sopta, J.,& Leposavić, G.. (2020). Supplementary information for the article: Dimitrijević, M.; Arsenović-Ranin, N.; Bufan, B.; Nacka-Aleksić, M.; Kosec, D.;  Pilipović, I.; Kotur-Stevuljević, J.; Simić, L.; Sopta, J.; Leposavić, G. Sex-Based Differences in Monocytic Lineage Cells  Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. Inflammation 2020, 43 (6),  2312–2331. https://doi.org/10.1007/s10753-020-01302-0.. in Inflammation
Springer., 43(6), 2312-2331.
https://hdl.handle.net/21.15107/rcub_intor_647

Dimitrijević M, Arsenović-Ranin N, Bufan B, Nacka-Aleksić M, Kosec D, Pilipović I, Kotur-Stevuljević J, Simić L, Sopta J, Leposavić G. Supplementary information for the article: Dimitrijević, M.; Arsenović-Ranin, N.; Bufan, B.; Nacka-Aleksić, M.; Kosec, D.;  Pilipović, I.; Kotur-Stevuljević, J.; Simić, L.; Sopta, J.; Leposavić, G. Sex-Based Differences in Monocytic Lineage Cells  Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. Inflammation 2020, 43 (6),  2312–2331. https://doi.org/10.1007/s10753-020-01302-0.. in Inflammation. 2020;43(6):2312-2331.
https://hdl.handle.net/21.15107/rcub_intor_647 .

Dimitrijević, Mirjana, Arsenović-Ranin, Nevena, Bufan, Biljana, Nacka-Aleksić, Mirjana, Kosec, Duško, Pilipović, Ivan, Kotur-Stevuljević, Jelena, Simić, Ljubica, Sopta, Jelena, Leposavić, Gordana, "Supplementary information for the article: Dimitrijević, M.; Arsenović-Ranin, N.; Bufan, B.; Nacka-Aleksić, M.; Kosec, D.;  Pilipović, I.; Kotur-Stevuljević, J.; Simić, L.; Sopta, J.; Leposavić, G. Sex-Based Differences in Monocytic Lineage Cells  Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. Inflammation 2020, 43 (6),  2312–2331. https://doi.org/10.1007/s10753-020-01302-0." in Inflammation, 43, no. 6 (2020):2312-2331,
https://hdl.handle.net/21.15107/rcub_intor_647 .

TY  - JOUR
AU  - Dimitrijević, Mirjana
AU  - Arsenović-Ranin, Nevena
AU  - Bufan, Biljana
AU  - Nacka-Aleksić, Mirjana
AU  - Kosec, Duško
AU  - Pilipović, Ivan
AU  - Kotur-Stevuljević, Jelena
AU  - Simić, Ljubica
AU  - Sopta, Jelena
AU  - Leposavić, Gordana
PY  - 2020
UR  - http://intor.torlakinstitut.com/handle/123456789/543
AB  - Monocytes' plasticity has an important role in the development of rheumatoid arthritis (RA), an autoimmune disease exhibiting greater prevalence in women. Contribution of this phenomenon to sex bias in RA severity was investigated in rat collagen-induced arthritis (CIA) model of RA. The greater severity of CIA in females (exhibiting signs of bone resorption) was accompanied by the higher blood level of advanced oxidation protein products and a more pro-oxidant profile. Consistently, in females, the greater density of giant multinuclear cells (monocytes/macrophages and osteoclasts) in inflamed joint tissue was found. This correlated with the higher frequencies of CCR2- and CX3CR1- expressing cells (precursors of inflammatory monocytes/macrophages and osteoclasts) among CD11b+ splenocytes. This in conjunction with the enhanced migratory capacity of CD11b+ monocytic cells in females compared with males could be linked with the higher frequencies of CCR2+CX3CR1-CD43(low)CD11b+ and CCR2-CX3CR1+CD43(hi)CD11b+ cells (corresponding to "classical" and "non-classical" monocytes, respectively) and the greater density of CD68+ cells (monocytes/macrophages and osteoclast precursors/osteoclasts) in blood and inflamed paws from female rats, respectively. Consistently, the higher levels of GM-CSF, TNF-alpha and IL-6, IL-1 beta (driving Th17 cell differentiation), and IL-17 followed by the lower level of IL-10 were measured in inflamed paw cultures from female compared with male rats. To the greater IL-17 production (associated with enhanced monocyte immigration and differentiation into osteoclasts) most likely contributed augmented Th17 cell generation in the lymph nodes draining arthritic joints from female compared with male rats. Overall, the study suggests the sex-specific contribution of monocytic lineage cells to CIA, and possibly RA development.
PB  - Springer/Plenum Publishers, New York
T2  - Inflammation
T1  - Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats
EP  - 2331
IS  - 6
SP  - 2312
VL  - 43
DO  - 10.1007/s10753-020-01302-0
ER  - 

@article{
author = "Dimitrijević, Mirjana and Arsenović-Ranin, Nevena and Bufan, Biljana and Nacka-Aleksić, Mirjana and Kosec, Duško and Pilipović, Ivan and Kotur-Stevuljević, Jelena and Simić, Ljubica and Sopta, Jelena and Leposavić, Gordana",
year = "2020",
abstract = "Monocytes' plasticity has an important role in the development of rheumatoid arthritis (RA), an autoimmune disease exhibiting greater prevalence in women. Contribution of this phenomenon to sex bias in RA severity was investigated in rat collagen-induced arthritis (CIA) model of RA. The greater severity of CIA in females (exhibiting signs of bone resorption) was accompanied by the higher blood level of advanced oxidation protein products and a more pro-oxidant profile. Consistently, in females, the greater density of giant multinuclear cells (monocytes/macrophages and osteoclasts) in inflamed joint tissue was found. This correlated with the higher frequencies of CCR2- and CX3CR1- expressing cells (precursors of inflammatory monocytes/macrophages and osteoclasts) among CD11b+ splenocytes. This in conjunction with the enhanced migratory capacity of CD11b+ monocytic cells in females compared with males could be linked with the higher frequencies of CCR2+CX3CR1-CD43(low)CD11b+ and CCR2-CX3CR1+CD43(hi)CD11b+ cells (corresponding to "classical" and "non-classical" monocytes, respectively) and the greater density of CD68+ cells (monocytes/macrophages and osteoclast precursors/osteoclasts) in blood and inflamed paws from female rats, respectively. Consistently, the higher levels of GM-CSF, TNF-alpha and IL-6, IL-1 beta (driving Th17 cell differentiation), and IL-17 followed by the lower level of IL-10 were measured in inflamed paw cultures from female compared with male rats. To the greater IL-17 production (associated with enhanced monocyte immigration and differentiation into osteoclasts) most likely contributed augmented Th17 cell generation in the lymph nodes draining arthritic joints from female compared with male rats. Overall, the study suggests the sex-specific contribution of monocytic lineage cells to CIA, and possibly RA development.",
publisher = "Springer/Plenum Publishers, New York",
journal = "Inflammation",
title = "Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats",
pages = "2331-2312",
number = "6",
volume = "43",
doi = "10.1007/s10753-020-01302-0"
}

Dimitrijević, M., Arsenović-Ranin, N., Bufan, B., Nacka-Aleksić, M., Kosec, D., Pilipović, I., Kotur-Stevuljević, J., Simić, L., Sopta, J.,& Leposavić, G.. (2020). Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. in Inflammation
Springer/Plenum Publishers, New York., 43(6), 2312-2331.
https://doi.org/10.1007/s10753-020-01302-0

Dimitrijević M, Arsenović-Ranin N, Bufan B, Nacka-Aleksić M, Kosec D, Pilipović I, Kotur-Stevuljević J, Simić L, Sopta J, Leposavić G. Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. in Inflammation. 2020;43(6):2312-2331.
doi:10.1007/s10753-020-01302-0 .

Dimitrijević, Mirjana, Arsenović-Ranin, Nevena, Bufan, Biljana, Nacka-Aleksić, Mirjana, Kosec, Duško, Pilipović, Ivan, Kotur-Stevuljević, Jelena, Simić, Ljubica, Sopta, Jelena, Leposavić, Gordana, "Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats" in Inflammation, 43, no. 6 (2020):2312-2331,
https://doi.org/10.1007/s10753-020-01302-0 . .