Browsing by Author "Mostarica Stojković, Marija (6701741422)"

Aim:To investigate the influences of betulinic acid (BA), a triterpenoid isolated from birch bark, on neuroinflammatory mediators involved in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis in vitro.Methods:Encephalitogenic T cells were prepared from draining lymph nodes and spinal cords of Dark Agouti rats 8 to 10 d after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Macrophages were isolated from the peritoneal cavity of adult untreated rats. Astrocytes were isolated from neonatal rat brains. The cells were cultured and then treated with different agents. IFN-γ, IL-17, iNOS and CXCL12 mRNA levels in the cells were analyzed with RT-PCR. iNOS and CXCL12 protein levels were detected using immunoblot. NO and ROS generation was measured using Griess reaction and flow cytometry, respectively.Results:In encephalitogenic T cells stimulated with MBP (10 μg/mL), addition of BA inhibited IL-17 and IFN-γ production in a dose-dependent manner. The estimated IC50 values for IL-17 and IFN γ were 11.2 and 63.8 μmol/L, respectively. When the macrophages were stimulated with LPS (10 ng/mL), addition of BA (50 μmol/L) significantly increased ROS generation, and suppressed NO generation. The astrocytes were stimulated with ConASn containing numerous inflammatory mediators, which mimicked the inflammatory milieu within CNS; addition of BA (50 μmol/L) significantly increased ROS generation, and blocked ConASn-induced increases in iNOS and CXCL12 mRNA levels, but did not affect iNOS and CXCL12 protein levels. Importantly, in both the macrophages and astrocytes, addition of BA (50 μmol/L) inhibited lipid peroxidation.Conclusion:Besides inhibiting encephalitogenic T cell cytokines and reducing NO generation, BA induces tissue-damaging ROS generation within CNS. © 2013 CPS and SIMM All rights reserved.

Aim:To investigate the influences of betulinic acid (BA), a triterpenoid isolated from birch bark, on neuroinflammatory mediators involved in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis in vitro.Methods:Encephalitogenic T cells were prepared from draining lymph nodes and spinal cords of Dark Agouti rats 8 to 10 d after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Macrophages were isolated from the peritoneal cavity of adult untreated rats. Astrocytes were isolated from neonatal rat brains. The cells were cultured and then treated with different agents. IFN-γ, IL-17, iNOS and CXCL12 mRNA levels in the cells were analyzed with RT-PCR. iNOS and CXCL12 protein levels were detected using immunoblot. NO and ROS generation was measured using Griess reaction and flow cytometry, respectively.Results:In encephalitogenic T cells stimulated with MBP (10 μg/mL), addition of BA inhibited IL-17 and IFN-γ production in a dose-dependent manner. The estimated IC50 values for IL-17 and IFN γ were 11.2 and 63.8 μmol/L, respectively. When the macrophages were stimulated with LPS (10 ng/mL), addition of BA (50 μmol/L) significantly increased ROS generation, and suppressed NO generation. The astrocytes were stimulated with ConASn containing numerous inflammatory mediators, which mimicked the inflammatory milieu within CNS; addition of BA (50 μmol/L) significantly increased ROS generation, and blocked ConASn-induced increases in iNOS and CXCL12 mRNA levels, but did not affect iNOS and CXCL12 protein levels. Importantly, in both the macrophages and astrocytes, addition of BA (50 μmol/L) inhibited lipid peroxidation.Conclusion:Besides inhibiting encephalitogenic T cell cytokines and reducing NO generation, BA induces tissue-damaging ROS generation within CNS. © 2013 CPS and SIMM All rights reserved.

Dendritic cells (DC) are responsible for the initiation and shaping of the adaptive immune response and are in the focus of autoimmunity research. We were interested in comparison of DC obtained from autoimmunity-prone Dark Agouti (DA) rats and autoimmunity-resistant Albino Oxford (AO) rats. DC were generated from bone marrow precursors and matured (mDC) by lipopolysaccharide. Tolerogenic DC (tolDC) obtained by vitamin D3 treatment were studied in parallel. Profile of cytokine production was different in AO and DA mDC and tolDC. Expression of MHC class II molecules and CD86 were higher in DA DC, while vitamin D3 reduced their expression in dendritic cells of both strains. Allogeneic proliferation of CD4+ T cells was reduced by AO tolDC, but not with DA tolDC in comparison to respective mDC. Finally, expression of various genes identified as differentially expressed in human mDC and tolDC was also analyzed in AO and DA DC. Again, AO and DA DC differed in the expression of the analyzed genes. To conclude, AO and DA DC differ in production of cytokines, expression of antigen presentation-related molecules and in regulation of CD4+ T proliferation. The difference is valuable for understanding the divergence of the strains in their susceptibility to autoimmunity. © 2019 Elsevier GmbH

Dendritic cells (DC) are responsible for the initiation and shaping of the adaptive immune response and are in the focus of autoimmunity research. We were interested in comparison of DC obtained from autoimmunity-prone Dark Agouti (DA) rats and autoimmunity-resistant Albino Oxford (AO) rats. DC were generated from bone marrow precursors and matured (mDC) by lipopolysaccharide. Tolerogenic DC (tolDC) obtained by vitamin D3 treatment were studied in parallel. Profile of cytokine production was different in AO and DA mDC and tolDC. Expression of MHC class II molecules and CD86 were higher in DA DC, while vitamin D3 reduced their expression in dendritic cells of both strains. Allogeneic proliferation of CD4+ T cells was reduced by AO tolDC, but not with DA tolDC in comparison to respective mDC. Finally, expression of various genes identified as differentially expressed in human mDC and tolDC was also analyzed in AO and DA DC. Again, AO and DA DC differed in the expression of the analyzed genes. To conclude, AO and DA DC differ in production of cytokines, expression of antigen presentation-related molecules and in regulation of CD4+ T proliferation. The difference is valuable for understanding the divergence of the strains in their susceptibility to autoimmunity. © 2019 Elsevier GmbH

Experimental autoimmune encephalomyelitis (EAE) is classically induced with complete Freund's adjuvant (CFA). The immune response against CFA has a confounding influence on the translational capacity of EAE as a multiple sclerosis model. Here, we compare clinical, cellular and molecular properties between syngeneic spinal cord homogenate (SCH)- and SCH + CFA-immunized Dark Agouti rats. EAE signs were observed earlier and the cumulative clinical score was higher without CFA. Also, a higher number of immune cells infiltrates in the spinal cords was noticed at the peak of EAE without CFA. High spinal cord abundance of CD8+CD11bc+MHC class II+ cells was detected in SCH-immunized rats. Myelin basic protein -specific response can be elicited in the cells from the lymph nodes draining the site of SCH immunization. This CFA-free EAE is a reliable multiple sclerosis model. © 2021 Elsevier B.V.

Experimental autoimmune encephalomyelitis (EAE) is classically induced with complete Freund's adjuvant (CFA). The immune response against CFA has a confounding influence on the translational capacity of EAE as a multiple sclerosis model. Here, we compare clinical, cellular and molecular properties between syngeneic spinal cord homogenate (SCH)- and SCH + CFA-immunized Dark Agouti rats. EAE signs were observed earlier and the cumulative clinical score was higher without CFA. Also, a higher number of immune cells infiltrates in the spinal cords was noticed at the peak of EAE without CFA. High spinal cord abundance of CD8+CD11bc+MHC class II+ cells was detected in SCH-immunized rats. Myelin basic protein -specific response can be elicited in the cells from the lymph nodes draining the site of SCH immunization. This CFA-free EAE is a reliable multiple sclerosis model. © 2021 Elsevier B.V.

CXCL12 plays a protective role in CNS autoimmunity. Expression of CXCL12-γ, which has distinct structural and functional properties than the other isoforms of CXCL12, was determined in spinal cords of rats immunized to develop experimental autoimmune encephalomyelitis (EAE). CNS expression of CXCL12-γ was markedly lower in EAE-prone Dark Agouti rats than in EAE-resistant Albino Oxford rats, both in spinal cord homogenates and micro-blood vessels isolated from spinal cords. Inhibition of nitric oxide (NO) synthesis in DA rats upregulated, while donation of NO in AO rats downregulated CNS expression of CXCL12-γ. NO inhibited CXCL12-γ expression in astrocytes in vitro. A splice variant of CXCL12-γ which migrates into nucleolus was not detected in spinal cord or astrocytes. Thus, CXCL12-γ is expressed in the CNS after EAE induction, but its expression is markedly suppressed in spinal cord affected with full blown inflammation. NO is an important regulator of CXCL12-γ expression in neuroinflammation. © 2013 Elsevier B.V.

CXCL12 plays a protective role in CNS autoimmunity. Expression of CXCL12-γ, which has distinct structural and functional properties than the other isoforms of CXCL12, was determined in spinal cords of rats immunized to develop experimental autoimmune encephalomyelitis (EAE). CNS expression of CXCL12-γ was markedly lower in EAE-prone Dark Agouti rats than in EAE-resistant Albino Oxford rats, both in spinal cord homogenates and micro-blood vessels isolated from spinal cords. Inhibition of nitric oxide (NO) synthesis in DA rats upregulated, while donation of NO in AO rats downregulated CNS expression of CXCL12-γ. NO inhibited CXCL12-γ expression in astrocytes in vitro. A splice variant of CXCL12-γ which migrates into nucleolus was not detected in spinal cord or astrocytes. Thus, CXCL12-γ is expressed in the CNS after EAE induction, but its expression is markedly suppressed in spinal cord affected with full blown inflammation. NO is an important regulator of CXCL12-γ expression in neuroinflammation. © 2013 Elsevier B.V.

Dark Agouti (DA) rats are highly susceptible to induction of experimental autoimmune encephalomyelitis (EAE), still they completely recover from the disease. Here, we were interested to determine contribution of major anti-inflammatory cytokines transforming growth factor (TGF)-β and interleukin (IL)-10 to the recovery of DA rats from EAE. To that extent we determined CNS expression of these cytokines in DA rats at different phases of EAE and compared data to those obtained in EAE-resistant Albino Oxford (AO) rats. Higher expression of TGF-β was persistently observed in the CNS of AO rats, even if rats were not immunized. This implied that high TGF-β within the CNS is important for resistance of AO rats to EAE induction. On the contrary, IL-10 expression was consistently higher in DA than in AO rats and it culminated at the peak of EAE. Methylprednisolone suppressed EAE and expression of IL-10 in spinal cord homogenates, while IL-10 was increased in CNS-infiltrating immune cells. This implied that IL-10 might have a significant role in recovery of DA rats from the disease. Thus, we next explored effects of IL-10 on astrocytes, glial cells that largely contribute to control of CNS inflammation. IL-10 stimulated astrocytic expression of an important regulator of neuroinflammation, CXCL12. Thus, IL-10 might contribute to recovery of DA rats from EAE through induction of CXCL12 expression in astrocytes. © 2013 Elsevier GmbH.

Dark Agouti (DA) rats are highly susceptible to induction of experimental autoimmune encephalomyelitis (EAE), still they completely recover from the disease. Here, we were interested to determine contribution of major anti-inflammatory cytokines transforming growth factor (TGF)-β and interleukin (IL)-10 to the recovery of DA rats from EAE. To that extent we determined CNS expression of these cytokines in DA rats at different phases of EAE and compared data to those obtained in EAE-resistant Albino Oxford (AO) rats. Higher expression of TGF-β was persistently observed in the CNS of AO rats, even if rats were not immunized. This implied that high TGF-β within the CNS is important for resistance of AO rats to EAE induction. On the contrary, IL-10 expression was consistently higher in DA than in AO rats and it culminated at the peak of EAE. Methylprednisolone suppressed EAE and expression of IL-10 in spinal cord homogenates, while IL-10 was increased in CNS-infiltrating immune cells. This implied that IL-10 might have a significant role in recovery of DA rats from the disease. Thus, we next explored effects of IL-10 on astrocytes, glial cells that largely contribute to control of CNS inflammation. IL-10 stimulated astrocytic expression of an important regulator of neuroinflammation, CXCL12. Thus, IL-10 might contribute to recovery of DA rats from EAE through induction of CXCL12 expression in astrocytes. © 2013 Elsevier GmbH.