Ceruloplasmin (Cp) is a ferroxidase that also plays a role in iron efflux from cells. It can thus help to regulate cellular iron homeostasis. In the CNS, Cp is expressed as a membrane-anchored form by astrocytes. Here, we assessed the role of Cp in permanent middle cerebral artery occlusion (pMCAO) comparing wildtype and Cp null mice. Our studies show that the lesion size is larger and functional recovery impaired in Cp null mice compared to wildtype mice. Expression of Cp increased ninefold at 72 h after pMCAO and remained elevated about twofold at day 14. We also assessed changes in mRNA and protein expression of molecules involved in iron homeostasis. As expected there was a reduction in ferroportin in Cp null mice at 72 h. There was also a remarkable increase in DMT1 protein in both genotypes at 72 h, being much higher in wildtype mice (19.5-fold), that then remained elevated about twofold at 14 days. No difference was seen in transferrin receptor 1 (TfR1) expression, except a small reduction in wildtype mice at 72 h, suggesting that the increase in DMT1 may underlie iron uptake independent of TfR1-endosomal uptake. There was also an increase of ferritin light chain in both genotypes. Iron histochemistry showed increased iron accumulation after pMCAO, initially along the lesion border and later throughout the lesion. Immunofluorescence labeling for ferritin (a surrogate marker for iron) and GFAP or CD11b showed increased ferritin in GFAP+ astrocytes along the lesion border in Cp null mice, while CD11b+ macrophages expressed ferritin equally in both genotypes. Increased lipid peroxidation assessed by 4HNE staining was increased threefold in Cp null mice at 72 h after pMCAO; and 3-nitrotyrosine labeling showed a similar trend. Three key pro-inflammatory cytokines (IL-1β, TNFα, and IL-6) were markedly increased at 24 h after pMCAO equally in both genotypes, and remained elevated at lower levels later, indicating that the lack of Cp does not alter key inflammatory cytokine expression after pMCAO. These data indicate that Cp expression is rapidly upregulated after pMCAO, and loss of Cp results in dysregulation of iron homeostasis, increased oxidative damage, greater lesion size and impaired recovery of function.
Astrocytes are the most abundant glial cells in the central nervous system (CNS) with the capacity to sense and react to injury and inflammatory events. While it has been widely documented that astrocytes can exert tissue-degenerative functions, less is known about their protective and disease-limiting roles. Here, we report the upregulation of pleiotrophin (PTN) by mouse and human astrocytes in multiple sclerosis (MS) and its preclinical model experimental autoimmune encephalomyelitis (EAE). Using CRISPR-Cas9-based genetic perturbation systems, we demonstrate in vivo that astrocyte-derived PTN is critical for the recovery phase of EAE and limits chronic CNS inflammation. PTN reduces pro-inflammatory signaling in astrocytes and microglia and promotes neuronal survival following inflammatory challenge. Finally, we show that intranasal administration of PTN during the late phase of EAE successfully reduces disease severity, making it a potential therapeutic candidate for the treatment of progressive MS, for which existing therapies are limited.
Canine hematopoietic progenitor cells were characterized by separation with monoclonal antibodies. Depleted and enriched fractions were studied for growth of CFU-GM in semisolid agar and for repopulating capacity of lethally irradiated dogs. CFU growth was not reduced by depletion of marrow using monoclonal antibodies F 3-20-7 (anti-dog Thy-1), MT606 (anti-human CD 6), and IOT2a (anti-human DR). CFU growth was variable following treatment with the anti-canine T-cell antibody MdT-P 1 and immunomagnetic bead separation. It was regularly enriched when MdT-P 1 treatment was followed by immunorosetting with staphylococcal protein A-loaded sheep red blood cells and density gradient separation. Lethally irradiated dogs were reconstituted by autologous marrow depleted of MdT-P 1-positive cells using immunorosetting and density gradient centrifugation, whereas immunomagnetic bead-depleted marrow was ineffective. Fluorescence-activated cell sorting showed enrichment of hematopoietic progenitor cells in the weakly MdT-P 1-positive fraction.
Recent advances in proteomic technologies now allow unparalleled assessment of the molecular composition of a wide range of sample types. However, the application of such technologies and techniques should not be undertaken lightly. Here, we describe why the design of a proteomics experiment itself is only the first step in yielding high-quality, translatable results. Indeed, the effectiveness and/or impact of the majority of contemporary proteomics screens are hindered not by commonly considered technical limitations such as low proteome coverage but rather by insufficient analyses. Proteomic experimentation requires a careful methodological selection to account for variables from sample collection, through to database searches for peptide identification to standardised post-mass spectrometry options directed analysis workflow, which should be adjusted for each study, from determining when and how to filter proteomic data to choosing holistic versus trend-wise analyses for biologically relevant patterns. Finally, we highlight and discuss the difficulties inherent in the modelling and study of the majority of progressive neurodegenerative conditions. We provide evidence (in the context of neurodegenerative research) for the benefit of undertaking a comparative approach through the application of the above considerations in the alignment of publicly available pre-existing data sets to identify potential novel regulators of neuronal stability.
The programmed cell death protein 1/programmed cell death ligand 1 axis plays an important role in the adaptive immune system and has influence on neoplastic and inflammatory diseases, while its role in multiple sclerosis is unclear. Here, we aimed to analyze expression patterns of programmed cell death protein 1 and programmed cell death ligand 1 on peripheral blood mononuclear cells and their soluble variants in multiple sclerosis patients and controls, to determine their correlation with clinical disability and disease activity. In a cross-sectional study, we performed in-depth flow cytometric immunophenotyping of peripheral blood mononuclear cells and analyzed soluble programmed cell death protein 1 and programmed cell death ligand 1 serum levels in patients with relapsing-remitting multiple sclerosis and controls. In comparison to control subjects, relapsing-remitting multiple sclerosis patients displayed distinct cellular programmed cell death protein 1/programmed cell death ligand 1 expression patterns in immune cell subsets and increased soluble programmed cell death ligand 1 levels, which correlated with clinical measures of disability and MRI activity over time. This study extends our knowledge of how programmed cell death protein 1 and programmed cell death ligand 1 are expressed in the membranes of patients with relapsing-remitting multiple sclerosis and describes for the first time the elevation of soluble programmed cell death ligand 1 in the blood of multiple sclerosis patients. The distinct expression pattern of membrane-bound programmed cell death protein 1 and programmed cell death ligand 1 and the correlation between soluble programmed cell death ligand 1, membrane-bound programmed cell death ligand 1, disease, and clinical factors may offer therapeutic potential in the setting of multiple sclerosis and might improve future diagnosis and clinical decision making.
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