I-AbACUS: a Reliable Software Tool for the Semi-Automatic Analysis of Invasion and Migration Transwell Assays

The quantification of invasion and migration is an important aspect of cancer research, used both in the study of the molecular processes involved in this collection of diseases and the evaluation of the efficacy of new potential treatments. The transwell assay, while being one of the most widely used techniques for the evaluation of these characteristics, shows a high dependence on the operator\u2019s ability to correctly identify the cells and a low protocol standardization. Here we present I-AbACUS, a software tool specifically designed to aid the analysis of transwell assays that automatically and specifically recognizes cells in images of stained membranes and provides the user with a suggested cell count. A complete description of this instrument, together with its validation against the standard analysis technique for this assay is presented. Furthermore, we show that I-AbACUS is versatile and able to elaborate images containing cells with different morphologies and that the obtained results are less dependent on the operator and their experience. We anticipate that this instrument, freely available (Gnu Public Licence GPL v2) at www.marilisacortesi.com as a standalone application, could significantly improve the quantification of invasion and migration of cancer cells

Antibodies to myelin oligodendrocyte glycoprotein have a demyelination phenotype in children and affect oligodendrocyte cytoskeleton

Objective: To examine the clinical features of paediatric CNS demyelination associated with positive myelin oligodendrocyte glycoprotein (MOG) antibodies, and examine the functional effects of MOG antibody on oligodendrocyte cytoskeleton. Methods: We measured MOG antibody using a flow cytometry live cell-based assay in acute sera of 73 children with CNS demyelination (DEM) (median age 8 years, range 1.3–15.3), followed for a median of 4 years. We used MO3.13 cells to examine IgG effects on oligodendrocyte cytoskeleton using 3-D deconvolution imaging. Results: MOG antibodies were found in 31/73 DEM (42%, n =73), but in 0/24 controls. At first presentation, MOG antibody-positive patients were more likely to have bilateral rather than unilateral optic neuritis (ON) (9/10 vs 1/5, respectively, p= 0.03), less likely to have brainstem findings (2/31 vs 16/42, p= 0.005), more likely to have a raised erythrocyte sedimentation rate N20 mm/h (9/19 vs 3/21, p= 0.05), less likely to have intrathecal oligoclonal band (0/16 v 5/27, p =0.18), and were less likely to be homozygous or heterozygous for HLA-DRB1*1501 (3/18 vs 7/22, p=0.46). MOG antibody positivity varied according to clinical phenotype, with ON and relapsing ON being most likely to be seropositive. Two relapsing MOG antibody-positive patients treated with mycophenolate mofetil remain in remission and have become MOG antibody seronegative. Oligodendrocytes incubated with purified IgG from MOG antibodypositive patients showed a striking loss of organization of the thin filaments and the microtubule cytoskeleton, as evidenced by F-actin and β-tubulin immunolabelings. Conclusions: MOG antibody may define a separate demyelination syndrome which has therapeutic implications. MOG antibody has functional effects on oligodendrocyte cytoskeleton.1 page(s

Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte cytoskeleton

Objective: To examine the clinical features of pediatric CNS demyelination associated with positive myelin oligodendrocyte glycoprotein (MOG) antibodies and to examine the functional effects of MOG antibody on oligodendrocyte cytoskeleton. Methods: We measured MOG antibody using a fluorescence-activated cell sorting live cell-based assay in acute sera of 73 children with CNS demyelination (DEM) (median age 8 years, range 1.3-15.3) followed for a median of 4 years. We used MO3.13 cells to examine immunoglobulin (Ig) G effects on oligodendrocyte cytoskeleton using 3D deconvolution imaging. Results: MOG antibodies were found in 31/73 patients with DEM (42%) but in 0/24 controls. At first presentation, MOG antibody-positive patients were more likely to have bilateral than unilateral optic neuritis (ON) (9/10 vs 1/5, respectively, p = 0.03), less likely to have brainstem findings (2/31 vs 16/42, p = 0.005), more likely to have a raised erythrocyte sedimentation rate >20 mm/h (9/19 vs 3/21, p = 0.05), less likely to have intrathecal oligoclonal bands (0/16 vs 5/27, p = 0.18), and less likely to be homozygous or heterozygous for human leukocyte antigen DRB1*1501 (3/18 vs 7/22, p = 0.46). MOG antibody positivity varied according to clinical phenotype, with ON and relapsing ON most likely to be seropositive. Two relapsing MOG antibody-positive patients treated with mycophenolate mofetil remain in remission and have become MOG antibody seronegative. Oligodendrocytes incubated with purified IgG from MOG antibody-positive patients showed a striking loss of organization of the thin filaments and the microtubule cytoskeleton, as evidenced by F-actin and β-tubulin immunolabelings. Conclusions: MOG antibody may define a separate demyelination syndrome, which has therapeutic implications. MOG antibody has functional effects on oligodendrocyte cytoskeleton.10 page(s