Extracellular Prion Protein Aggregates in Nine Gerstmann–Sträussler–Scheinker Syndrome Subjects with Mutation P102L: A Micromorphological Study and Comparison with Literature Data

Gerstmann–Sträussler–Scheinker syndrome (GSS) is a hereditary neurodegenerative disease characterized by extracellular aggregations of pathological prion protein (PrP) forming characteristic plaques. Our study aimed to evaluate the micromorphology and protein composition of these plaques in relation to age, disease duration, and co-expression of other pathogenic proteins related to other neurodegenerations. Hippocampal regions of nine clinically, neuropathologically, and genetically confirmed GSS subjects were investigated using immunohistochemistry and multichannel confocal fluorescent microscopy. Most pathognomic prion protein plaques were small (2–10 µm), condensed, globous, and did not contain any of the other investigated proteinaceous components, particularly dystrophic neurites. Equally rare (in two cases out of nine) were plaques over 50 µm having predominantly fibrillar structure and exhibit the presence of dystrophic neuritic structures; in one case, the plaques also included bulbous dystrophic neurites. Co-expression with hyperphosphorylated protein tau protein or amyloid beta-peptide (Aβ) in GSS PrP plaques is generally a rare observation, even in cases with comorbid neuropathology. The dominant picture of the GSS brain is small, condensed plaques, often multicentric, while presence of dystrophic neuritic changes accumulating hyperphosphorylated protein tau or Aβ in the PrP plaques are rare and, thus, their presence probably constitutes a trivial observation without any relationship to GSS development and progression.

Extracellular Protein Aggregates Colocalization and Neuronal Dystrophy in Comorbid Alzheimer’s and Creutzfeldt–Jakob Disease: A Micromorphological Pilot Study on 20 Brains

Alzheimer’s disease (AD) and sporadic Creutzfeldt–Jakob disease (sCJD) are both characterized by extracellular pathologically conformed aggregates of amyloid proteins—amyloid β-protein (Aβ) and prion protein (PrPSc), respectively. To investigate the potential morphological colocalization of Aβ and PrPSc aggregates, we examined the hippocampal regions (archicortex and neocortex) of 20 subjects with confirmed comorbid AD and sCJD using neurohistopathological analyses, immunohistochemical methods, and confocal fluorescent microscopy. Our data showed that extracellular Aβ and PrPSc aggregates tended to be, in most cases, located separately, and “compound” plaques were relatively rare. We observed PrPSc plaque-like structures in the periphery of the non-compact parts of Aβ plaques, as well as in tau protein-positive dystrophic structures. The AD ABC score according to the NIA-Alzheimer’s association guidelines, and prion protein subtype with codon 129 methionine–valine (M/V) polymorphisms in sCJD, while representing key characteristics of these diseases, did not correlate with the morphology of the Aβ/PrPSc co-aggregates. However, our data showed that PrPSc aggregation could dominate during co-aggregation with non-compact Aβ in the periphery of Aβ plaques.

TMIC-50. 3D QUANTIFICATION OF DYNAMIC CYTOKINE GRADIENTS PRODUCED BY PRIMARY TUMOR MODELS

BACKGROUND & SIGNIFICANCE The immune response is a coordinated effort directed by cytokine gradients and concentrations. High sensitivity and spatial resolution are necessary to resolve cytokine gradients in 3-dimensions. This system uses in situ confocal fluorescent microscopy with printed bead-based immunoassays. The combination of 3D printing of the beads and biofabrication of patient derived tumors allows for direct imaging, quantification, and movies of tumor cytokine secretion in response to challenges from immune cells, tumor associated fibroblasts, and chemotherapeutic agents. HYPOTHESIS: Cytokine dynamics can be measured in real-time by balancing concentrations of detection antibodies in solution with stationary immunoassay beads with measured capture and release rates. Favorable balancing of assay kinetics can facilitate measurements of concentrations of cytokines between 50 pg/mL to over 2,000 pg/mL. METHODS In situ confocal fluorescent microscopy identifies position, fluorescence, and type of bead relative to the in vitro tumor. Cytokines including IL2, IL6, IL8, IL11, and IFNg and growth factors such as VEGF and TGFb have been measured for a wide range of solid tumors (brain, sarcoma, and epithelial) under different stresses. RESULTS Dynamic gradients of IL8 were found across multiple tumor models showing local concentrations in excess of 2,000 pg/mL. Production rates were estimated to be over 1 protein per second for each cell, and inclusion of cancer associated support cells showed 10x increases in some cytokines. CD4+ cell production of IL2 was confirmed and quantified and showed strong sensitivity to tumor activity and antigen presentation. Multiplexed beads of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 allow for simultaneous measurement of multiple chemokines. Uncertainties associated with fluorescence measurement and quantification of concentrations will be discussed. CONCLUSIONS This integrated system of 3D assay printing, biofabrication of tumors and immune cell constructs, and in situ confocal microscopy provides the first direct measurements of 3D cytokine gradients in response to tumor stress.

A novel hybrid blend based on phenoxy-substituted boron subphthalocyanine for organic photodetectors

Phenoxy-substituted boron subphthalocyanine, blended with a conductive polymer MEH-PPV, is presented as a photoresistive organic material. Using an easily accessible drop casting technique, the blend produces a thin-layer organic photoresistor with a photoresistive ratio of ~2–12. Variations in the blend composition and morphology are shown to change the transport properties of the material. The photoelectrochemical characteristics of the photoresistor are discussed in terms of impedance spectroscopy and the morphology of the material is analyzed using confocal fluorescent microscopy. The device developed is a daylight detector.

Cisplatin Fails To Stimulate Production Of Reactive Oxygen Species and Release Of Neutrophil Extracellular Traps By Human Neutrophils In Vitro

Background Cisplatin is a commonly used antineoplastic agent for treatment of a broad range of cancers. Cisplatin-based treatment has been associated with a significant risk of venous thromboembolism. The mechanisms through which cisplatin contributes to a prothrombotic state remain unclear. Neutrophil extracellular traps (NETs) consist of web-like DNA–histone core decorated with granule proteins and are released from activated neutrophils in a process dependent on reactive oxygen species (ROS), in particular hypochlorous acid (HOCl). Recently, NETs have been shown to play an important role in initiation and propagation of venous thrombus in a number of animal models of deep vein thrombosis. The aim of this study was to investigate whether NETs may provide a potential link between cisplatin and venous thromboembolism. Methods and Results To assess the effect of cisplatin on release of NETs by ex vivo human neutrophils isolated by positive immunomagnetic selection we visualised NETs release by confocal fluorescent microscopy and performed fluorimetric quantification of cell-free DNA (CFDNA) using either SYTOX Green nucleic acid stain (10 µM) or an ultrasensitive fluorescent assay Picogreen Quant IT (Invitrogen). In contrast to stimulation with phorbol 12-myristate 13-acetate (PMA) (25 nM),which resulted in 22 ng/104neutrophils of detectable CFDNA, neither of these two assays could detect any significant release of CFDNA by human neutrophils exposed to cisplatin (15 µM) for 2 or 4 hours above baseline similar with vehicle control. Furthermore, confocal fluorescent microscopy imaging of neutrophils stained with non-cell permeable DNA dye SYTOX Red (Invitrogen) demonstrated no difference in NET formation between control and cisplatin treated human neutrophils. Thus we could not demonstrate that NETS are produced in response to cisplatin treatment. In view of consistent reports that NET formation is ROS dependent we decided to investigate whether cisplatin exposure leads to production of ROS by human neutrophils. Few published studies into the effects of cisplatin on the production of ROS by human neutrophils in vitro offer conflicting results. We used flow cytometry and fluorescent probe hydroethidine (HE) for detection of intercellular superoxide anion radical in HL60 granulocytic cells in the presence of cisplatin (up to 50 µM). Differentiation down the granulocytic lineage after stimulation with ATRA was confirmed by light microscopy and by flow cytometry. Capacity of differentiated HL60 cells to generate NET formation after PMA stimulation was confirmed by fluorescence microscopy. Cisplatin failed to augment the spontaneous production of ROS by ATRA differentiated HL60 cells. The number of viable ethidium-high cells in cisplatin treated group did not differ from the vehicle control indicating no detectable production of ROS in response to cisplatin. In contrast, positive control treatment with PMA (25 nM) and menadione (40 µM) resulted in 4- and 20-fold increase in viable ethidium-high population respectively. ROS generation by human neutrophils was measured by a colorimetric assay for chlorination of extracellular taurine to determine if exposure to cisplatin results in the production of HOCl by human neutrophils in vitro. Treatment of resting neutrophils with cisplatin (15 µM) for 30 min or 120 min was not associated with an increase in the spontaneous production of HOCl above the baseline. Furthermore, the PMA (25 nM)-activated generation of HOCl production was not increased by pre-treating neutrophils with cisplatin indicating that there was no potentiation of ROS by pre-treatment with cisplatin. Discussion and Conclusion Our results suggest that cisplatin fails to induce release of NETs or HOCl from human neutrophils in vitro. These negative findings seem to be at odds with the well described pro-oxidative actions of cisplatin. One possible explanation centres on reported findings that the pro-oxidative effects of cisplatin are dependent on the mitochondrial generation of ROS whilst the mitochondria-generated ROS appear not to be instrumental to NET formation. Therefore, we postulate that cisplatin may not be able to induce NET formation by human neutrophils, which are known to contain few mitochondria, due to a sub-threshold ROS signal. Therefore it appears that cisplatin-associated increased risk of venous thrombosis is unlikely to be mediated through NETs. Disclosures: No relevant conflicts of interest to declare.