Effective Antigen-Specific Immunotherapy in the Marmoset Model of Multiple Sclerosis

Mature T cells initially respond to Ag by activation and expansion, but high and repeated doses of Ag cause programmed cell death and can suppress T cell-mediated diseases in rodents. We evaluated repeated systemic Ag administration in a marmoset model of experimental allergic encephalomyelitis that closely resembles the human disease multiple sclerosis. We found that treatment with MP4, a chimeric, recombinant polypeptide containing human myelin basic protein and human proteolipid protein epitopes, prevented clinical symptoms and did not exacerbate disease. CNS lesions were also reduced as assessed in vivo by magnetic resonance imaging. Thus, specific Ag-directed therapy can be effective and nontoxic in primates. The Journal of Immunology, 2001, 166: 2116 -2121. M ultiple sclerosis (MS) 4 is a paralytic disease involving destruction of myelin sheaths surrounding axons in the CNS (1, 2). MS affects young adults, most often women residing in northern latitudes. The disease exhibits relapsing and remitting symptoms including disturbances in vision, speech, coordination, and cognition as well as weakness, spasticity, and paralysis (1, 2). Lymphocytic infiltration in the CNS white matter and immune reactions against myelin Ags indicate an autoimmune etiology for MS (1-8). Allergic encephalomyelitis was first observed as a side effect of the rabies vaccine prepared from rabbit brains by Pasteur in the 1880s (see Ref. 3). Rivers and others showed that the CNS inflammation was caused not by the rabies virus but by immune sensitization to the combination of adjuvant and brain tissue contaminating the vaccine (3, 4). Experimental allergic encephalomyelitis (EAE) models in various animal species, typically rodents, were later developed by immunization with myelin proteins in adjuvant or by the adoptive transfer of myelinreactive T cells, causing inflammatory damage to the white matter (1-6). Rodent EAE is the most widely used disease model despite important differences from MS (2). Encephalitogenic CD4 ϩ T cells are believed to initiate and perpetuate EAE and MS and thus constitute a therapeutic target (1-8). Abundant myelin protein Ags, including myelin basic protein (MBP) and proteolipid protein (PLP) as well as the less abundant Ags, myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG), are recognized by T cells in MS patients (9 -11). T cell responses against MBP and PLP may occur at an increased frequency in MS patients compared with controls (1, 2, 11, 12). Ag-specific immunotherapies directed at T cells could avoid the harmful side effects of general immunosuppressive treatments. We have investigated a potential immunotherapy for MS based on our observation that T cells undergo apoptosis both in vitro and in vivo when exposed to high or repeated doses of their cognate Ag (13, To present a broad array of potential epitopes to reactive T cells, we constructed MP4, a protein chimera of the 21.5-kDa isoform of human MBP, and a modified form of human PLP, termed PLP4, that lacks the hydrophobic domains of the protein but includes all of the known T cell epitopes (19 -21). MP4 is processed into multiple determinants and can eliminate rodent EAE by promoting tolerance to different epitopes In a few instances, EAE and Ag treatments have been studied in nonhuman primates. EAE was originally induced in rhesus macaques using CNS homogenates or purified MBP (3, 4, 30 -32). It was also found that repeated injections of MBP could arrest EAE The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

A Connection between Colony Biomass and Death in Caribbean Reef-Building Corals

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994–2007), eleven years in the Exuma Cays, Bahamas (1995–2006), and four years in Puerto Morelos, Mexico (2003–2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1–4 m) compared to deeper-dwelling conspecifics (12–15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD

<div><p>Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in <i>trans</i>) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in <i>cis</i>). As a result, forms of DeltaD that have the MID can activate Notch in <i>trans</i> and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in <i>trans</i> fails but ability to inhibit in <i>cis</i> is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.</p></div

Endocytosis of DeltaD deletion mutants.

<p>(A,E,I,M,Q,U) Distribution of zdd2 (green) in COS7 cells transfected with DeltaD (A) or DeltaD ∆A, ∆B, ∆C, ∆D or ∆A-D deletion mutants (E, I, M, Q, U). Surface DeltaD was first labelled by incubation with zdd2 at 4°C for 30’ then, following washout of unbound zdd2, internalization was allowed for 30’ at 37°C. Nuclei were labelled with DAPI (blue). (B-D, F-H, J-L, N-P, R-T, V-X) Distribution of zdd2 (green) in COS7 cells co-transfected with DeltaD constructs and Mib1 (red) following internalization as described above. Each set of 3 panels, respectively, shows distribution of the DeltaD construct (green), Myc-Mib1 (red)/nuclei (blue), and the merged image. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127864#sec002" target="_blank">materials and methods</a> for details.</p

Identification of critical residues in the Mib1-Interacting Domain (MID).

<p>(A) Conserved amino acids (pink shading) in the putative <i>Mib1</i>-Interacting Domain in Delta ligands. (B) KK and NN/KK mutants do not significantly interact with mind bomb. DeltaD-HA, ∆B and point mutation (NN, KK and NN/KK) constructs are co-immunoprecipitated with Myc-Mib1 using anti-Myc Ab and detected with anti-HA Ab. (C) KK and NN/KK mutants are not significantly ubiquitylated by Mib1. DeltaD-HA, ∆B and point mutation constructs co-transfected with Flag-Ubiquitin (Flag-Ub) with and without Myc-Mib1 are immunoprecipitated with anti-HA Ab and detected with Anti-Flag Ab to assay ubiquitylation of full length and mutant forms of DeltaD.</p