Author Of 2 Presentations
P0087 - Identification of proteins associated with ageing in patients with progressive multiple sclerosis (ID 1589)
Abstract
Background
Similar to other neurodegenerative disorders, the onset of progressive MS is related to age, a factor known to amplify neurodegeneration. Recent studies have shown that exposure of aged mice to a young blood circulation through parabiosis or administration of young blood plasma (plasma from 3-month-old mice) reverses cognitive deficits observed with normal ageing.
Objectives
We aimed to search for soluble factors in the serum of patients with progressive MS that are affected by age and are differentially decreased in patients compared to healthy controls (HC) of similar age.
Methods
Protein levels were determined in serum samples from a cohort of 30 untreated MS patients (15 patients with secondary progressive MS - SPMS - and 15 with primary progressive MS - PPMS) and 25 HC. Progressive MS patients were classified according to age and clinical characteristics into the following three groups (each group containing 10 patients, 5 with SPMS and 5 with PPMS): (i) 40 ± 3 years old, disease duration <10 years, EDSS <4.5; (ii) 50 ± 3 years old, disease duration between 10-20 years, EDSS between 4.5-6; and (iii) 60 ± 3 years old, disease duration >20 years, EDSS >6.5. HC were classified based on age into the following groups (each group containing 5 individuals): 20, 30, 40, 50, and 60 ± 3 years old. To maximize the breadth and depth of serum proteome coverage, the top 70 abundant proteins in serum were depleted. Afterwards, samples were subjected to mass spectrometry.
Results
After depletion of the most abundant proteins in serum, a total of 2,059 molecules were detected in all 55 samples. The maSigPro package (R Bioconductor) was used to identify proteins with significantly divergent expression profiles as a function of time. A quadratic regression model was fit for each molecule and 823 proteins, among the 2059 analyzed, were found differentially expressed (FDR < 0.05) between the MS group and HC. The serum levels of the following proteins were significantly decreased by ageing in progressive MS patients compared with HC and were selected for further studies: PLXDC2, Neudesin, Myostatin, Myocilin, and EMMPRIN.
Conclusions
Protein expression profiling associated with ageing in progressive MS patients and HC lead to the identification of number of promising candidates associated with neurotrophic functions, myelination, and nervous system development. Results obtained by mass spectrometry need to be validated by targeted immunoassays.
P0330 - Effects of cladribine on proliferation, survival and cytokine release of human astrocytes (ID 1581)
Abstract
Background
Cladribine is a synthetic purine nucleoside analogue with immunosuppressive functions that has demonstrated beneficial effects in patients with relapsing-remitting multiple sclerosis (MS) and that may also regulate the immune function as an analogue of adenosine receptors. Although the effect of cladribine is well studied on immune cells, it remains unveiled how it affects the immune function of glial populations of the central nervous system.
Objectives
In the context of MS, we aimed to test the effect of cladribine on proliferation, survival and cytokine release of human astrocytes.
Methods
To assess the effect of cladribine on cell survival and proliferation, primary human astrocytes were cultured with cladribine at high concentrations (2µM, 0.2µM), at the mean estimated brain exposure of the drug (0.02µM) and at a low concentration (0.002µM) for 72h. The percentages of dead and proliferating cells were determined by flow cytometry. To assess the effect of cladribine on cytokine release, human astrocytes were stimulated for 6h with 20ng/ml IL1-β and TNF-α. The stimulus was withdrawn and cells were cultured for additional 18h. Cladribine was added for the whole 24h of culture. Supernatants were harvested to quantify IL1-β, IL6, TNF-α and GM-CSF release by Luminex. To assess the effect of cladribine independently of deoxycytidine kinase (DCK), deoxycytidine was also added to human astrocytes.
Results
Only high concentrations of cladribine induced death on human astrocytes (2µM: 35.89%±7.62 or 0.2µM: 7.27%±3.12 vs control: 3.17%1.84±; p<0.0001 and p=0.156, respectively) and inhibited their proliferative capacity (2µM: 0.96%±1.14 or 0.2µM: 14.06%±5.44 vs control: 33.06%±1.42; both p-values<0.0001). Additionally, the percentage of proliferating cells in the 2µM and 0.2µM conditions presented a limited capacity of proliferation (measured as the Relative Intensity of Proliferation Staining respect to basal; 2µM: 0.24±0.04 and 0.2µM: 0.55±0.22, both p-values<0.0001). When DCK activity was blocked by deoxycytidine, cell death and proliferation were reversed to control condition values. We are currently determining the effect of cladribine on pro-inflammatory cytokine release by human astrocytes.
Conclusions
The mean estimated brain exposure to cladribine does not influence cell survival or proliferation of human astrocytes neither in a DCK-dependent nor in a DCK-independent manner, suggesting that cladribine does not affect the normal astrocyte function in MS patients.
Presenter Of 1 Presentation
P0087 - Identification of proteins associated with ageing in patients with progressive multiple sclerosis (ID 1589)
Abstract
Background
Similar to other neurodegenerative disorders, the onset of progressive MS is related to age, a factor known to amplify neurodegeneration. Recent studies have shown that exposure of aged mice to a young blood circulation through parabiosis or administration of young blood plasma (plasma from 3-month-old mice) reverses cognitive deficits observed with normal ageing.
Objectives
We aimed to search for soluble factors in the serum of patients with progressive MS that are affected by age and are differentially decreased in patients compared to healthy controls (HC) of similar age.
Methods
Protein levels were determined in serum samples from a cohort of 30 untreated MS patients (15 patients with secondary progressive MS - SPMS - and 15 with primary progressive MS - PPMS) and 25 HC. Progressive MS patients were classified according to age and clinical characteristics into the following three groups (each group containing 10 patients, 5 with SPMS and 5 with PPMS): (i) 40 ± 3 years old, disease duration <10 years, EDSS <4.5; (ii) 50 ± 3 years old, disease duration between 10-20 years, EDSS between 4.5-6; and (iii) 60 ± 3 years old, disease duration >20 years, EDSS >6.5. HC were classified based on age into the following groups (each group containing 5 individuals): 20, 30, 40, 50, and 60 ± 3 years old. To maximize the breadth and depth of serum proteome coverage, the top 70 abundant proteins in serum were depleted. Afterwards, samples were subjected to mass spectrometry.
Results
After depletion of the most abundant proteins in serum, a total of 2,059 molecules were detected in all 55 samples. The maSigPro package (R Bioconductor) was used to identify proteins with significantly divergent expression profiles as a function of time. A quadratic regression model was fit for each molecule and 823 proteins, among the 2059 analyzed, were found differentially expressed (FDR < 0.05) between the MS group and HC. The serum levels of the following proteins were significantly decreased by ageing in progressive MS patients compared with HC and were selected for further studies: PLXDC2, Neudesin, Myostatin, Myocilin, and EMMPRIN.
Conclusions
Protein expression profiling associated with ageing in progressive MS patients and HC lead to the identification of number of promising candidates associated with neurotrophic functions, myelination, and nervous system development. Results obtained by mass spectrometry need to be validated by targeted immunoassays.