Contributions
Abstract: EP1453
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Background: Adoptively transferred experimental autoimmune encephalomyelitis (at-EAE) is an animal model that allows for a better delineation of the effector phase of EAE, regardless of the inductor. There is no consensus about an ideal protocol in the existing literature.
Objectives: In order to develop a more severe animal model of at-EAE, encephalytogenic cells were tested in vitro to study different conditions of stimulation before they were transferred to recipient animals.
Methods: Splenocytes and lymph nodes from C57BL/6mice with actively induced EAE were cultured in different conditions with MOG35-55 (25 or 50µg/ml), IL-12 (25ng/ml) and with or without IL-18 (25ng/ml) at 0, 60, 72 and 96 hours. Differential activation of the encephalytogenic cells was studied by detecting the following markers: CD69, IL-2 and IFN-γ by flow cytometry.
An in vivo experiment was conducted in C57BL/6 mice, using the two experimental conditions that produced more cell activation. The animals were evaluated for clinical scores, and the expression of axonal damage (SMI32), demyelination (MBP), astroglial response (GFAP) and microglia activation(Iba1), were determined in spinal cords by confocal microscopy.
Results: Concentration of MOG35-55, cytokine combination and time of culture were decisive in activation/cell death. The optimal condition of culture was 25µg/ml MOG + 25ng/ml IL-12 + 25ng/ml IL-18 during 72 hours. In this condition, coculture of IL-18 and Il-12 induced higher levels of IFN-γ, compared with IL-12 alone . At 96 hours, the activation was greater, but also the death of the cells, as it was the case using 50 µg/ml MOG35-55.
In the in vivo experiment, the progress of clinical signs was homogeneous, they started at the same time and reached a higher score in the IL-18 group.
Axonal damage and demyelination were increased in spinal cords of mice when IL-18 was added in at-EAE induction. No differences were found in astroglial response and microglial activation.
Conclusions: IL-18 increases the clinical severity of at-EAE. An optimal boosting of encephalytogenic cells in vitro might be achieved with the combination of IL-12 and IL-18, which induces a significant secretion of IFN-γ.
Disclosure: Antonio García Merino: has received honoraria for lecturing, consulting or travel expenses from Bayer, Biogen-Idec, Merck, Teva, Novartis, Roche, Almirall and Genzyme, and research grants from Merck and Novartis.
Ruth García Hernández: has received honoraria for lecturing and travel expenses from Teva, Novartis, and Genzyme.
Irene Moreno Torres: has received honoraria for lecturing and travel expenses from Merck, Teva, Novartis, Roche and Genzyme.
The other authors do not have any conflicts of interest to report.
Abstract: EP1453
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Background: Adoptively transferred experimental autoimmune encephalomyelitis (at-EAE) is an animal model that allows for a better delineation of the effector phase of EAE, regardless of the inductor. There is no consensus about an ideal protocol in the existing literature.
Objectives: In order to develop a more severe animal model of at-EAE, encephalytogenic cells were tested in vitro to study different conditions of stimulation before they were transferred to recipient animals.
Methods: Splenocytes and lymph nodes from C57BL/6mice with actively induced EAE were cultured in different conditions with MOG35-55 (25 or 50µg/ml), IL-12 (25ng/ml) and with or without IL-18 (25ng/ml) at 0, 60, 72 and 96 hours. Differential activation of the encephalytogenic cells was studied by detecting the following markers: CD69, IL-2 and IFN-γ by flow cytometry.
An in vivo experiment was conducted in C57BL/6 mice, using the two experimental conditions that produced more cell activation. The animals were evaluated for clinical scores, and the expression of axonal damage (SMI32), demyelination (MBP), astroglial response (GFAP) and microglia activation(Iba1), were determined in spinal cords by confocal microscopy.
Results: Concentration of MOG35-55, cytokine combination and time of culture were decisive in activation/cell death. The optimal condition of culture was 25µg/ml MOG + 25ng/ml IL-12 + 25ng/ml IL-18 during 72 hours. In this condition, coculture of IL-18 and Il-12 induced higher levels of IFN-γ, compared with IL-12 alone . At 96 hours, the activation was greater, but also the death of the cells, as it was the case using 50 µg/ml MOG35-55.
In the in vivo experiment, the progress of clinical signs was homogeneous, they started at the same time and reached a higher score in the IL-18 group.
Axonal damage and demyelination were increased in spinal cords of mice when IL-18 was added in at-EAE induction. No differences were found in astroglial response and microglial activation.
Conclusions: IL-18 increases the clinical severity of at-EAE. An optimal boosting of encephalytogenic cells in vitro might be achieved with the combination of IL-12 and IL-18, which induces a significant secretion of IFN-γ.
Disclosure: Antonio García Merino: has received honoraria for lecturing, consulting or travel expenses from Bayer, Biogen-Idec, Merck, Teva, Novartis, Roche, Almirall and Genzyme, and research grants from Merck and Novartis.
Ruth García Hernández: has received honoraria for lecturing and travel expenses from Teva, Novartis, and Genzyme.
Irene Moreno Torres: has received honoraria for lecturing and travel expenses from Merck, Teva, Novartis, Roche and Genzyme.
The other authors do not have any conflicts of interest to report.