- and Alexa 680 and Phaloidin Alexa Fluor 546 were from Life Technologies (Carlsbad, CA). Myelin oligodendrocyte glycoprotein (MOG) was purchased from Anaspec (Fremont, CA) and Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA
T helper type 17 lymphocytes (Th17 cells) infiltrate the central nervous system (CNS), induce inflammation and demyelination and play a pivotal role in the pathogenesis of multiple sclerosis (MS). Sialomucin CD43 is highly expressed in Th17 cells and mediates adhesion to endothelial selectin (E-selectin), an initiating step in Th17 cell recruitment to sites of inflammation. CD43-/- mice have impaired Th17 cell recruitment to the CNS and are protected from Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of multiple sclerosis. However, E-selectin is dispensable for the development of EAE, in contrast to intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). We report that CD43-/- mice have decreased demyelination and T cell infiltration, but similar upregulation of ICAM-1 and VCAM-1 in the spinal cord, as compared to WT mice, at the initiation of EAE. CD43-/- Th17 cells have impaired adhesion to ICAM-1 under flow conditions in vitro, despite having similar expression of LFA-1, the main T cell ligand for ICAM-1, as WT Th17 cells. Regardless of the route of integrin activation, CD43-/- Th17 cells firm arrest on ICAM-1 was comparable to WT Th17 cells, but CD43-/- Th17 cells failed to optimally apically migrate on immobilized ICAM-1 coated coverslips and endothelial cells, and to transmigrate under shear flow conditions in an ICAM-1 dependent manner. Collectively, these findings unveil novel roles for CD43, facilitating adhesion of Th17 cells to ICAM-1 and modulating apical and transendothelial migration, as mechanisms potentially responsible for Th17 cell recruitment to sites of inflammation such as the CNS. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Therapeutic Potential of Conditioned Medium Derived from Oligodendrocytes Cultured in a Self-Assembling Peptide Nanoscaffold in Experimental Autoimmune Encephalomyelitis
- The mice also received ip injection of pertussis toxin (300 ng in 100 μl PBS; List Biological Lab, USA) at the time of immunization and again 48 hours later.(Majidi-Zolbanin et al., 2015) For dose-response studies, C57BL/6 mice were randomly divided into four groups
Normal human urothelial cells were grown to confluence in complete keratinocyte serum-free medium (cKSFM) with human recombinant epithelial growth factor (5 ng/mL) bovine, pituitary extract (50 mg/mL; Gibco), supplemented with cholera toxin (30 ng/mL; List Biological Laboratories, Campbell, CA, USA) under standard conditions.
Hemocyanins are oxygen-transporting glycoproteins in molluscs and arthropods. In this study, we assayed the biomacromolecules from the molluscs Helix lucorum (HlH), Rapana venosa (RvH) and Megatura crenulata (KLH) including their functional units (FUs), for therapy of bladder cancer permanent cells. In vitro studies antitumour activities of these proteins were performed with bladder cancer permanent cell line CAL-29 and the normal urothelial cell line HL 10/29 in comparison to doxorubicin. The obtained results showed that the human tumour CAL-29 cell line is sensitive to the action of the tested hemocyanins and their isoforms. We observed a dose- and time-dependent inhibition of tumour cell growth after incubation with native HlH and two FUs (βc-HlH-a and FU βc-HlH-h); and of particular significance, FU βc-HlH-h showed a surprisingly stronger effect than that the doxorubicin-treated cells. Cells treated with βc-HlH-h, showed both, apoptotic and necrotic cells. In addition, two-dimensional polyacrylamide gel electrophoresis (PAGE) found for differential up-regulation of several proteins after hemocyanin treatment. Eight different down-regulated and two up-regulated proteins were identified, which may be associated with the apoptosis pathway. No inhibition of the normal urothelial cell line HL 10/29 was observed after treatment with HlH and its isoforms. The most effective inhibition of CAL-29 tumour cells was observed after treatment with βc-HlH-h, probably caused by a specific oligosaccharide structure of HlH with methylated hexoses. These results suggest that hemocyanin glycosylation plays an important role in its anticancer activity.
A Novel Supplementation Approach to Enhance Host Response to Sublingual Vaccination
... lutions were added to J774 macrophages cultured in cultured in RPMI supplemented with 10% fetal calf serum. Bacillus anthracis lethal toxin (LeTx) [i.e., PA plus Bacillus anthracis lethal factor (LF, List Biological, Campbell, CA)] was then added to the plates. After overnight incubation, MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added to assess the ... lutions were added to J774 macrophages cultured in cultured in RPMI supplemented with 10% fetal calf serum. Bacillus anthracis lethal toxin (LeTx) [i.e., PA plus Bacillus anthracis lethal factor (LF, List Biological, Campbell, CA)] was then added to the plates. After overnight incubation, MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added to assess the
Sublingual immunization is emerging as an alternative to nasal immunization and induction of mucosal IgA responses. Using Bacillus anthracis edema toxin (EdTx) as an adjuvant, we previously showed that innate responses triggered after sublingual immunization could limit generation of IgA responses. We tested whether co-administration of a neutrophil elastase inhibitor (NEI) could rescue the ability of EdTx to induce broad antibody responses, including mucosal IgA. NEI supplementation of sublingual vaccines containing EdTx promoted antigen-specific serum IgA responses but also enhanced serum IgG1, and IgG2b responses. This enhancing effect of NEI did not extend to all antibody isotypes and IgG sublclasses, since NEI reduced serum IgE responses and did not affect IgG2a/c and IgG3 responses. NEI supplementation also promoted anti-Bacillus anthracis protective antigen (PA) neutralizing antibodies and enhanced high affinity IgG1 and IgA antibodies. In addition to serum IgA, NEI supplementation stimulated antigen-specific mucosal IgA responses in the GI tract, and enhanced antigen-specific IgG responses in vaginal washes. Analysis of CD4+ T helper cell responses revealed that co-administration of NEI broadened the profile of cytokine responses, by stimulating Th1, Th2, Th17, and Tfh cytokines. We also noted that NEI had a higher stimulatory effect on IL-5, IL-10, IL-17 responses.
Neuron-specific PERK inactivation exacerbates neurodegeneration during experimental autoimmune encephalomyelitis
... ptide emulsified in complete Freund’s adjuvant (BD Biosciences) supplemented with 600 μg _Mycobacterium tuberculosis_ (strain H37Ra; BD Biosciences). Two i.p. injections of 400 ng pertussis toxin (List Biological Laboratories) were given 0 and 48 hours later. Clinical scores (0, healthy; 1, flaccid tail; 2, ataxia and/or paresis of hind limbs; 3, paralysis of hind limbs and/or paresis of foreli ... ptide emulsified in complete Freund’s adjuvant (BD Biosciences) supplemented with 600 μg _Mycobacterium tuberculosis_ (strain H37Ra; BD Biosciences). Two i.p. injections of 400 ng pertussis toxin (List Biological Laboratories) were given 0 and 48 hours later. Clinical scores (0, healthy; 1, flaccid tail; 2, ataxia and/or paresis of hind limbs; 3, paralysis of hind limbs and/or paresis of foreli
Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are chronic inflammatory demyelinating and neurodegenerative diseases of the CNS. Although neurodegeneration is the major contributor to chronic disability in MS, mechanisms governing the viability of axons and neurons in MS and EAE remain elusive. Data indicate that activation of pancreatic endoplasmic reticulum kinase (PERK) influences, positively or negatively, neuron and axon viability in various neurodegenerative diseases through induction of ATF4. In this study, we demonstrate that the PERK pathway was activated in neurons during EAE. We found that neuron-specific PERK inactivation impaired EAE resolution and exacerbated EAE-induced axon degeneration, neuron loss, and demyelination. Surprisingly, neuron-specific ATF4 inactivation did not alter EAE disease course or EAE-induced axon degeneration, neuron loss, and demyelination. These results suggest that PERK activation in neurons protects axons and neurons against inflammation in MS and EAE through ATF4-independent mechanisms.