Immuno-analysis of microparticles: Probing at the limits of detection

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ∼60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data

Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice.

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases

Topoisomerase I inhibition leads to length-dependent gene expression changes in human primary astrocytes

Topoisomerase I is required for the proper expression of long genes (> 100 kb) in mouse and human cortical neurons, including many candidate genes for autism spectrum disorder (ASD) [1]. Given the important role of astrocytes in brain development [2], we investigated whether long genes, including autism susceptibility genes, also require topoisomerase I expression in human primary astrocytes. We carried genome-wide expression profiling of cultured human primary astrocytes following treatment with the topoisomerase I inhibitor Topotecan, using Illumina microarrays. We identified several thousands of differentially expressed genes and confirmed that topoisomerase I inhibition affects gene expression in human primary astrocytes in a length-dependent manner. We also identified over 20 ASD-associated genes that show topoisomerase-dependent gene expression in human primary astrocytes but have not been previously reported as topoisomerase-I-dependent in neurons. The microarray data have been deposited in NCBI GEO (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE90052