Development and Preclinical Evaluation of a Copper-64-Labeled Antibody Targeting Glycine-Alanine Dipeptides for PET Imaging of C9orf72-Associated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia.

Aggregating poly(glycine-alanine) (poly-GA) is derived from the unconventional translation of the pathogenic intronic hexanucleotide repeat expansion in the C9orf72 gene, which is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly-GA accumulates predominantly in neuronal cytoplasmic inclusions unique to C9orf72 ALS/FTD patients. Poly-GA is, therefore, a promising target for PET/CT imaging of FTD/ALS to monitor disease progression and therapeutic interventions. A novel 64Cu-labeled anti-GA antibody (mAb1A12) targeting the poly-GA protein was developed and evaluated in a transgenic mouse model. It was obtained with high radiochemical purity (RCP), radiochemical yield (RCY), and specific activity, and showed high stability in vitro and ex vivo and specifically bound to poly-GA. The affinity of NODAGA-mAb1A12 for poly-GA was not affected by this modification. [64Cu]Cu-NODAGA-mAb1A12 was injected into transgenic mice expressing GFP-(GA)175 in excitatory neurons driven by Camk2a-Cre and in control littermates. PET/CT imaging was performed at 2, 20, and 40 h post-injection (p.i.) and revealed a higher accumulation in the cortex in transgenic mice than in wild-type mice, as reflected by higher standardized uptake value ratios (SUVR) using the cerebellum as the reference region. The organs were isolated for biodistribution and ex vivo autoradiography. Autoradiography revealed a higher cortex-to-cerebellum ratio in the transgenic mice than in the controls. Results from autoradiography were validated by immunohistochemistry and poly-GA immunoassays. Moreover, we confirmed antibody uptake in the CNS in a pharmacokinetic study of the perfused tissues. In summary, [64Cu]Cu-NODAGA-mAb1A12 demonstrated favorable in vitro characteristics and an increased relative binding in poly-GA transgenic mice compared to wild-type mice in vivo. Our results with this first-in-class radiotracer suggested that targeting poly-GA is a promising approach for PET/CT imaging in FTD/ALS.

Activated mast cells increase the level of endothelin-1 mRNA in cocultured endothelial cells and degrade the secreted Peptide.

Subendothelial mast cells have been implicated in the pathogenesis of allergic inflammation, in atherosclerosis, and in the regulation of vascular tone. Because endothelin-1 (ET-1) is an important regulator of vascular tone and has also been implicated in the pathogenesis of atherosclerosis, we studied the role of mast cells in the metabolism of endothelial cell-derived ET-1. In mast cell-endothelial cell cocultures, activation of the mast cells with ensuing degranulation was accompanied by the increased expression of ET-1 mRNA in the endothelial cells, yet the immunoreactive ET-1 protein in the coculture medium disappeared almost completely during the 24-hour coculture. Activation of the mast cells with the ensuing degranulation resulted in proteolytic degradation of ET-1 by the 2 neutral proteases, chymase and carboxypeptidase A, of the exocytosed mast cell granules. With synthetic ET-1 and purified mast cell granule enzymes, efficient degradation of ET-1 by chymase and carboxypeptidase A was verified. These in vitro results imply a novel role for mast cell-derived neutral proteases in ET-1 metabolism and suggest that activated subendothelial mast cells are important local regulators of ET-1 metabolism.

TRAC in high-content gene expression analysis: applications in microbial population studies, process biotechnology and biomedical research.

More than a decade of intensive use of microarray technology has flooded the scientific community with genome-wide expression data of diverse biological states. As a result, connection of the expression signatures of a relatively small number of genes related to, for example, disease states, patient responses or toxicological responses has become possible. Development of tools that enable cost- and time-efficient analysis of such signatures from large sample numbers is currently of major interest for research, drug screening and diagnostic purposes. A method named transcript analysis with aid of affinity capture (TRAC) is a novel solution hybridization and bead-based assay enabling multiplex mRNA target detection simultaneously from large sample numbers. Functionality of TRAC has been shown in a number of applications, including microbial quantification, gene expression-based monitoring of biotechnical processes, cell-based cancer marker gene screening and siRNA validation, which are reviewed here.

Receptors for the anaphylatoxins C3a and C5a are expressed in human atherosclerotic coronary plaques.

The anaphylatoxins C3a and C5a are potent chemotactic and pro-inflammatory peptides that are released during complement activation, and recent clinical work have suggested them a role in acute coronary events. Here we studied whether human coronary plaques express anaphylatoxin receptors C3aR and C5aR, i.e. whether they have the potential to respond to anaphylatoxins. For this purpose, both normal (n=14) and atherosclerotic (n=20) human coronary artery samples were collected for histological and PCR analyses. Immunohistochemistry demonstrated that in atherosclerotic, but not in normal intimas, C3aR and C5aR were present. Consistently, PCR analysis showed that the expression of both receptors was >5-fold increased in the atherosclerotic plaques (p<0.01). Double immunofluorescence stainings revealed that in the plaques the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR and a small fraction of them also expressed C3aR, the mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. In conclusion, the presence of receptors for anaphylatoxins in human coronary plaques suggests that anaphylatoxins activate coronary plaques, and points the complement system as a potential therapeutic target in attempts to stabilize them.