Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased Calpain and caspase activity and can be reduced by erythropoietin treatment

The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation. These events cause blood–brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF) signaling pathway have been shown. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine CM with or without the use of erythropoietin (EPO) as adjunct therapy. ELISA and western blotting was used for quantification of VEGF and relevant proteins in brain and plasma. CM increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. EPO treatment normalized VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF)-1α was significantly upregulated whereas cerebral HIF-2α and EPO levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in EPO-treated mice. Also caspase and calpain activity was reduced markedly in EPO-treated mice

An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells

Introduction: Reporter cells expressing a chimeric receptor that activates a reporter can be used for screening ligand-mediated signal transduction. In this study, we used reporter cells harboring an NFAT/lacZ construct that express β-galactosidase when the chimeric receptor is stimulated. A colorimetric β-galactosidase substrate, chlorophenol-red β-d-galactopyranoside (CPRG), was used to detect enzymatic activity. Sub-optimal conditions have unfortunately extensively been reported with such reporter-based β-galactosidase assays. Here, we aimed to improve the CPRG-based colorimetric assay such that receptor ligands could be effectively screened with reporter cells. Methods: After stimulation of reporter cells, we determined β-galactosidase activity by absorbance measurement of β-galactosidase-dependent CPRG hydrolysis. We systematically examined each component in a standard lysis buffer most commonly reported for this type of reporter cells. Furthermore, we evaluated literature in the field. Results: An increased CPRG substrate concentration combined with a different detergent, Saponin, and an optimal wavelength recording markedly increased the sensitivity for the detection of β-galactosidase activity (≈ 4-fold increase). Moreover, the improved protocol resulted in increased linear time-dependent recording of enzymatic activity once cells had been lysed, and a more stable and reproducible assay to detect a ligand-stimulus with the reporter cells. The optimal time length of exposure to a stimulus was ligand-dependent. Discussion: In conclusion, we provide an improved protocol with an optimized lysis buffer that gives up to a six-fold higher and more robust specific signal when NFAT/lacZ-based receptor-expressing reporter cells are exposed to a stimulus

Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased calpain and caspase activity and can be reduced by erythropoietin treatment

The pathogenesis of cerebral malaria includes compromised microvascular perfusion, increased inflammation, cytoadhesion and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and can be associated with the vascular endothelial growth factor (VEGF) signalling pathway. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine cerebral malaria with or without the use of erythropoietin as adjunct therapy. ELISA and western blotting was used for quantification of VEGF and relevant proteins in brain and plasma. Cerebral malaria increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. Erythropoietin treatment normalised VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF)-1α was significantly upregulated whereas cerebral HIF-2α and erythropoietin levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in erythropoietin-treated mice. Also caspase and calpain activity was reduced markedly in erythropoietin-treated mice

Analyzing microglial-associated Aβ in Alzheimer’s disease transgenic mice with a novel mid-domain Aβ-antibody

The mechanisms of amyloid-β (Aβ)-degradation and clearance in Alzheimer’s disease (AD) pathogenesis have been relatively little studied. Short Aβ-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aβ-antibody raised against an Aβ mid-domain and used it to investigate microglial Aβ-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aβ-mid-domain antibody (ab338), raised against the mid-domain amino acids 21–34 (Aβ21–34), was characterized with biochemical and histological techniques. To identify the epitope in Aβ recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10−10 M) and showed preference for C-terminal truncated Aβ-peptides ending at amino acid 34 and Aβ-mid domain peptides with high scores of β-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aβ-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aβ-clearance in vivo. The ab338 antibody might be a valuable tool to study Aβ-clearance by microglial uptake and Aβ-mid-domain peptides generated by enzymatic degradation and alternate production