Explore the vast range of titles available.

We have previously reported evidence that Nogo-A activation of Nogo-receptor 1 (NgR1) can drive axonal dystrophy during the neurological progression of experimental autoimmune encephalomyelitis (EAE). However, the B-cell activating factor (BAFF/BlyS) may also be an important ligand of NgR during neuroinflammation. In the current study we define that NgR1 and its homologs may contribute to immune cell signaling during EAE. Meningeal B-cells expressing NgR1 and NgR3 were identified within the lumbosacral spinal cords of ngr1 +/+ EAE-induced mice at clinical score 1. Furthermore, increased secretion of immunoglobulins that bound to central nervous system myelin were shown to be generated from isolated NgR1- and NgR3-expressing B-cells of ngr1 +/+ EAE-induced mice. In vitro BAFF stimulation of NgR1- and NgR3-expressing B cells, directed them into the cell cycle DNA synthesis phase. However, when we antagonized BAFF signaling by co-incubation with recombinant BAFF-R, NgR1-Fc, or NgR3 peptides, the B cells remained in the G0/G1 phase. The data suggest that B cells express NgR1 and NgR3 during EAE, being localized to infiltrates of the meninges and that their regulation is governed by BAFF signaling.

Myelin-associated inhibitory factors within the central nervous system (CNS) are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1 (NgR1) has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis (MS). In this study, the animal model of MS, experimental autoimmune encephalomyelitis (EAE) was induced in ngr1+/+ and ngr1-/- female mice following injection with the myelin oligodendrocyte glycoprotein (MOG35-55) peptide. A fate-map analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively (increasing locomotor disability) from both genotypes, using the CD11b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1+/+ and ngr1-/- mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1+/+ and ngr1-/- mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1+/+ and ngr1-/- mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of NgR on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1-/- mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1-/- mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1-/- mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.

Axo-glial units are highly organised microstructures propagating saltatory conduction and are disrupted during multiple sclerosis (MS). Nogo receptor 1 (NgR1) has been suggested to govern axonal damage during the progression of disease in the MS-like mouse model, experimental autoimmune encephalomyelitis (EAE). Here we have identified that adult ngr1 −/− mice, previously used in EAE and spinal cord injury experiments, display elongated paranodes, and nodes of Ranvier. Unstructured paranodal regions in ngr1 −/− mice are matched with more distributed expression pattern of Caspr. Compound action potentials of optic nerves and spinal cords from naïve ngr1 −/− mice are delayed and reduced. Molecular interaction studies revealed enhanced Caspr cleavage. Our data suggest that NgR1 may regulate axo-myelin ultrastructure through Caspr-mediated adhesion, regulating the electrophysiological signature of myelinated axons of central nervous system (CNS).

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles. Prior to the development of these characteristic pathological hallmarks of AD, anterograde axonal transport is impaired. However, the key proteins that initiate these intracellular impairments remain elusive. The collapsin response mediator protein-2 (CRMP-2) plays an integral role in kinesin-1-dependent axonal transport and there is evidence that phosphorylation of CRMP-2 releases kinesin-1. Here, we tested the hypothesis that amyloid-beta (Aβ)-dependent phosphorylation of CRMP-2 disrupts its association with the kinesin-1 (an anterograde axonal motor transport protein) in AD. We found that brain sections and lysates from AD patients demonstrated elevated phosphorylation of CRMP-2 at the T555 site. Additionally, in the transgenic Tg2576 mouse model of familial AD (FAD) that exhibits Aβ accumulation in the brain with age, we found substantial co-localization of pT555CRMP-2 and dystrophic neurites. In SH-SY5Y differentiated neuronal cultures, Aβ-dependent phosphorylation of CRMP-2 at the T555 site was also elevated and this reduced the CRMP-2 association with kinesin-1. The overexpression of an unphosphorylatable form of CRMP-2 in neurons promoted the re-establishment of CRMP-2-kinesin association and axon elongation. These data suggest that Aβ-dependent phosphorylation of CRMP-2 at the T555 site may directly impair anterograde axonal transport protein function, leading to neuronal defects.

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.

House dust mite (HDM) is the commonest allergen trigger globally for allergic rhinitis and atopic asthma. To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect allergen sensitization using the flow cytometric CytoBas assay. Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1 and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 HDM-allergic patients and 26 non-HDM-sensitized controls were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. The tetramers effectively bound and activated basophils from allergic patients but not non-sensitized controls. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%), similarly for CytoBas with a single Der p 2 (95% and 96%) to BAT (95% and 87%) in detecting allergen sensitization. A positive staining for Der p 1 and/or Der p 2 was 100% sensitive and 96% specific for HDM allergy. CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to a BAT assay, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis. A single flow cytometry stain of basophils (CytoBas) with both Der p 1 and Der p 2 provides >95% specificity and sensitivity for detection of functional HDM allergen sensitization. Fluorescent tetramers of recombinant Der f 1, Der p 1 and Der p 2 can be used to detect functional IgE sensitization to house dust mite (HDM) by flowcytometric detection on basophils (CytoBas). A single CytoBas assay with inclusion of both Der p 1 and Der p 2 can detect HDM sensitization with >95% sensitivity and specificity.