Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials

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Quantification of parenchymal calcifications in chronic pancreatitis: relation to atrophy, ductal changes, fibrosis and clinical parameters

OBJECTIVES: Parenchymal calcifications are considered a hallmark finding of chronic pancreatitis (CP), but little is known about its relation to the clinical presentation and other morphological features such as atrophy, fibrosis and ductal changes. The aim was to quantify the number and maximal size of parenchymal calcifications assessed on computed tomography (CT) and to explore the association with other CT and magnetic resonance imaging (MRI)-based pancreatic features and clinical parameters.METHODS: A well-characterised cohort of 54 CP patients was included. CT measurements included number and size of parenchymal calcifications, gland diameter and ductal diameter. MRI measurements included gland volume, ductal diameter, fibrosis (diffusion) and fatty infiltration (Dixon). Clinical parameters included body mass index (BMI), CP duration and aetiology, M-ANNHEIM clinical stage, tobacco use, alcohol consumption, the presence of diabetes, faecal elastase, clinical pain score and quality of life.RESULTS: There were no correlations between the number and size of parenchymal calcifications and any of the other morphological CT and MRI parameters (all p &gt; .05), except for larger size of calcifications in patients with high number of calcifications (p &lt; .001). The number of parenchymal calcifications was negatively correlated with BMI (r = -0.35, p = .0088). The number and size of parenchymal calcifications did not correlate with any of the other clinical parameters (all p &gt; .2).CONCLUSION: Our findings could indicate the existence of parenchymal calcifications as an independent pathophysiological process involved in the development of CP. Translational impact: Quantifications of calcifications could, in combination with other imaging biomarkers, be a useful imaging marker relevant for characterising CP.</p

Decellularised Human Umbilical Artery as a Vascular Graft Elicits Minimal Pro-Inflammatory Host Response Ex Vivo and In Vivo

Small diameter (&lt;6 mm) vessel grafts still pose a challenge for scientists worldwide. Decellularised umbilical artery (dUA) remains promising as small diameter tissue engineered vascular graft (TEVG), yet their immunogenicity remains unknown. Herein, we evaluated the host immune responses, with a focus on the innate part, towards human dUA implantation in mice, and confirmed our findings in an ex vivo allogeneic human setup. Overall, we did not observe any differences in the number of circulating white blood cells nor the number of monocytes among three groups of mice (1) dUA patch; (2) Sham; and (3) Mock throughout the study (day −7 to 28). Likewise, we found no difference in systemic inflammatory and anti-inflammatory cytokine levels between groups. However, a massive local remodelling response with M2 macrophages were observed in the dUA at day 28, whereas M1 macrophages were less frequent. Moreover, human monocytes from allogeneic individuals were differentiated into macrophages and exposed to lyophilised dUA to maximize an eventual M1 response. Yet, dUA did not elicit any immediate M1 response as determined by the absence of CCR7 and CXCL10. Together this suggests that human dUA elicits a minimal pro-inflammatory response further supporting its use as a TEVG in an allogeneic setup

The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study

Spinal cord injury (SCI) initiates detrimental cellular and molecular events that lead to acute and delayed neuroinflammation. Understanding the role of the inflammatory response in SCI requires insight into the temporal and cellular synthesis of inflammatory mediators. We subjected C57BL/6J mice to SCI and investigated inflammatory reactions. We examined activation, recruitment, and polarization of microglia and infiltrating immune cells, focusing specifically on tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2. In the acute phase, TNF expression increased in glial cells and neuron-like cells, followed by infiltrating immune cells. TNFR1 and TNFR2 levels increased in the delayed phase and were found preferentially on neurons and glial cells, respectively. The acute phase was dominated by the infiltration of granulocytes and macrophages. Microglial/macrophage expression of Arg1 increased from 1–7 days after SCI, followed by an increase in Itgam, Cx3cr1, and P2ry12, which remained elevated throughout the study. By 21 and 28 days after SCI, the lesion core was populated by galectin-3+, CD68+, and CD11b+ microglia/macrophages, surrounded by a glial scar consisting of GFAP+ astrocytes. Findings were verified in postmortem tissue from individuals with SCI. Our findings support the consensus that future neuroprotective immunotherapies should aim to selectively neutralize detrimental immune signaling while sustaining pro-regenerative processes