Noel G. Carlson

Treatment of multiple sclerosis with an anti–interleukin-2 receptor monoclonal antibody

We examined whether treatment with daclizumab, a humanized monoclonal antibody specific for the interleukin‐2 receptor α chain, was safe and efficacious in relapsing–remitting and secondary progressive multiple sclerosis patients. Nineteen ambulatory patients with clinically active disease were treated for 5 to 25 months. Seventeen patients were not responding to other immunotherapies. Daclizumab was generally well tolerated. Sustained clinical improvement (10 patients) or stabilization (9 patients) was observed. Daclizumab treatment produced significant reduction in magnetic resonance imaging activity. Ann Neurol 2004

Daclizumab phase II trial in relapsing and remitting multiple sclerosis: MRI and clinical results

Objective: Daclizumab is an interleukin 2 receptor α chain specific humanized monoclonal antibody that has shown promising therapeutic effects in multiple sclerosis (MS). Daclizumab treatment in patients with relapsing and remitting MS was administered to determine effects on MRI and clinical outcomes. Methods: Patients with MS on interferon (IFN) therapy but with continuing relapses and contrast enhancing lesions (CEL) were selected. Patients were evaluated with monthly MRI scans and clinical rating scales starting 3 months prior to treatment and then at 0.5 to 27.5 months during treatment. Daclizumab (1 mg/kg IV) was administered twice in the first month (initiated and administered again in 2 weeks), followed by treatments every 4 weeks. IFN was continued until 5.5 months after daclizumab was initiated. Patients were then placed on daclizumab monotherapy. Patients with recurrent CEL were restarted on IFN with daclizumab therapy at (1.5 mg/kg IV) every 28 days. Results: Nine patients qualified for inclusion and completed the trial. Efficacy measured by both total CEL and new CEL (p < 0.001), relapses, timed ambulation, Expanded Disability Status Scale, and Neurologic Rating Scale (p < 0.05 to p < 0.01) was observed. Conclusion: Daclizumab was effective in reducing contrast enhancing lesions and improving clinical scores in patients with relapsing and remitting multiple sclerosis with active disease not controlled by interferon therapy. These results provide evidence for long-term efficacy and support further clinical development of daclizumab.

Cutting Edge: Granzyme B Proteolysis of a Neuronal Glutamate Receptor Generates an Autoantigen and Is Modulated by Glycosylation

Autoimmune processes are initiated when tolerance to self-proteins fails to be established or maintained and immune cells are stimulated by self-Ags. Although intracellular autoantigens are common, the origin of extracellular autoantigens is poorly defined and possibly more dangerous. In this study, we considered a mechanism for the origin of an extracellular autoantigen from the neuronal glutamate receptor subunit 3 (GluR3) in Rasmussen’s encephalitis, a severe form of pediatric epilepsy. We demonstrate that specific cleavage of GluR3 by granzyme B (GB), a serine protease released by activated immune cells, can generate the GluR3B autoantigenic peptide, but only if an internal N-linked glycosylation sequon within the GluR3-GB recognition sequence (ISND*S) is not glycosylated. However, this N-glycon sequon while glycosylated normally is inefficiently used and glycosylation can fail. These results suggest that GB/N-glycon sites may escape normal tolerance mechanisms and contribute to autoantibody-mediated immune diseases.

Nicotine blocks TNF-α-mediated neuroprotection to NMDA by an α-bungarotoxin-sensitive pathway

Excitotoxic neuronal death mediated by N-methyl-D-aspartate (NMDA) glutamate receptors can contribute to the extended brain damage that often accompanies trauma or disease. Both the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and nicotine have been identified as possible neuroprotective agents to NMDA assault. We find that TNF-alpha protection of a subpopulation of cultured cortical neurons to chronic NMDA-mediated excitotoxic death requires both the activation of the p55/TNFRI, but not p75/TNFRII, and the release of endogenous TNF-alpha. Nicotine protection to NMDA was mediated through an alpha-bungarotoxin-sensitive receptor. When coapplied, neuroprotection to NMDA by either TNF-alpha or nicotine was abolished but could be recovered with alpha-bungarotoxin. These results suggest that the cytokine TNF-alpha and alpha-bungarotoxin-sensitive nicotinic neurotransmitter receptors confer neuroprotection through potentially antagonistic pathways.

Neuronal nicotinic acetylcholine receptor expression by O2A/oligodendrocyte progenitor cells

Oligodendrocyte precursor cells (O2A/OPC, A2B5+) were examined for expression of neuronal nicotinic acetylcholine receptors (nAChR). RT‐PCR analysis and immunocytochemistry of O2A/OPCs purified from the rat corpus collusum revealed the expression of nAChR subunits α3, α4, α5, α7, β2, and β4. Immunoreactivity toward nAChR subunits was not detected in cells induced to differentiate into either oligodendrocytes or astrocytes. Approximately 65% of O2A/OPCs loaded with the calcium‐responsive dye FURA‐2 increased their intracellular free calcium in response to nicotine application. This response was sensitive to the nAChRα4/β2 antagonist, dihydro‐β‐erythroidine (DHβE), and the voltage‐gated calcium channel antagonist, nifedipine. A subset of nicotine‐responsive cells (37%) established DHβE or nifedipine‐sensitive intracellular free calcium oscillations that continued in the presence of nicotine. Typical oscillations occurred at intervals of 20 to 30 s with progressively diminished amplitudes over a period of 2 to 3 min. In rare cases, oscillations persisted for as long as 10 min. O2A/OPCs exposed to carbachol or AMPA produced no oscillations despite robust increases in intracellular free calcium. The expression of nAChRs in non‐neuronal glial precursor cells suggests an expanded role for this receptor system in the development of the mammalian brain. GLIA 33:306–313, 2001. Published 2001 Wiley‐Liss, Inc.

Neuroprotection of cultured cortical neurons mediated by the cyclooxygenase‐2 inhibitor APHS can be reversed by a prostanoid

The neuroprotective properties of two cyclooxygenase‐2 (COX‐2) specific inhibitors, N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl]‐methanesulfonamide (NS398) and o‐(acetoxy‐phenyl)hept‐2‐ynyl2 sulfide (APHS) were examined in vitro using a mixed cortical neuronal culture system. Each of these inhibitors conferred a concentration‐dependent neuroprotective effect against an excitotoxic assault induced by NMDA. Neuroprotection was observed when the COX‐2 inhibitor was added before or even 1–3 hours after NMDA, which was coincident with an NMDA‐induced increase of COX‐2 transcripts in neurons. To test whether these COX‐2 inhibitors confer neuroprotection by inhibiting biosynthesis of prostanoids that may contribute toward excitotoxicity, two NMDA‐induced prostanoids, PGE2 and PGF2α, were tested for their ability to reverse the neuroprotective properties of APHS. APHS‐mediated neuroprotection was overcome by the concentration‐dependent (as low as 100 nM) administration of a synthetic analog of PGE2, 17‐phenyl‐trinor‐PGE2 (17‐pt‐E2), which is a relatively specific agonist for the EP1 and EP3 prostaglandin receptors; however, PGF2α had no significant effect on neuroprotection conferred by APHS. In the absence of APHS, neuroprotection was observed with either prostanoid. PGE2 may in some instances contribute toward excitotoxicity, and the inhibition of synthesis of this prostanoid may in part explain the neuroprotective properties of these COX‐2 inhibitors. Published 2002 Wiley‐Liss, Inc.

Neuronal expression of tumor necrosis factor alpha in the murine brain.

We have examined the expression of the inflammatory cytokine, tumor necrosis factor alpha (TNF alpha) in the mouse brain. Using immunohistochemical methods developed, we found anti-TNF alpha immunoreactivity localized in the basal ganglia and other discrete brain structures. Constitutive immunoreactivity, present in normal, unstimulated brain, was observed in glial and microglial-like cells, but it was predominant in neuronal-like cells. Intravenous administration of lipopolysaccharide (LPS) increased TNF alpha transcript levels detected by RT-PCR in specific brain subregions in which contaminating blood cells were removed. The maximal increase occurred within 2 h of LPS injection; transcripts diminished to near control levels in the next 4 h. Immunocytochemical analysis and single-cell RT-PCR analysis of primary cultures of cortical neuronal cells confirmed expression of TNF alpha in cells that also express neuronal-specific enolase RNA. Addition of LPS or recombinant TNF alpha protein to neuronal cultures enhanced expression of TNF alpha transcripts. Our results indicate that in addition to glial and microglial cells, a well-defined subset of neuronal cells also express TNF alpha constitutively; this expression can be altered by both extrinsic (LPS) and intrinsic (TNF alpha itself) factors.

Antioxidants in multiple sclerosis: Do they have a role in therapy?

Multiple sclerosis (MS) is an immune-mediated disease, with inflammation and neurodegeneration contributing to neuronal demyelination and axonal injury. Current therapies for MS are directed toward modulation of the immune response; however, there is increasing evidence that oxidative stress is an important component in the pathogenesis of MS.The inflammatory environment in demyelinating lesions is conducive to the generation of reactive oxygen species. When these species are generated in MS and animal models of MS, products such as peroxynitrite and Superoxide are formed that are highly toxic to cells. There are several examples of potential beneficial effects from various antioxidants in animal models of MS, but the efficacy may vary between different agents and, in some instances, may yield deleterious effects. Despite these promising results in animal models, there is limited and conflicting evidence of potential therapeutic effects of antioxidants such as vitamins C and E in treating MS. However, clinical trials in MS patients with more potent antioxidants, identified in animal studies, have been initiated.

Identification of Amino Acids in the Glutamate Receptor, GluR3, Important for Antibody-binding and Receptor-specific Activation

We reported (Twyman, R. E., Gahring, L. C., Speiss, J., and Rogers, S. W. (1995) Neuron 14, 755-762) that antibodies to a subregion of the glutamate receptor (GluR) subunit GluR3 termed GluR3B (amino acids 372-395), act as highly specific GluR agonists. In this study we produced additional rabbit anti-GluR3B-specific antibodies, ranked them according to their ability to function as GluR agonists and characterized the immunoreactivity using deletion and alanine substitution mutagenesis. These anti-GluR3B antibodies bound to a subset of the residues in GluR3B (amino acids 372-386), of which glutamate 375, valine 378, proline 379, and phenylalanine (Phe) 380 were preferred. The level of GluR activation correlated with the binding of antibody to Phe-380, which suggests that immunoreactivity directed toward Phe-380 is an index for the anti-GluR agonist potential. Since the identity of this residue varies between respective GluR subunits, this suggested that this residue may be important for imparting antibody subunit specificity. To test this possibility, the alanine in GluR1 was converted to a phenylalanine, which extended the subunit specificity from GluR3 to the modified GluR1. We conclude that antibody contacts with key residues in the GluR3B region define a novel GluR subunit-specific agonist binding site and impart subunit-specific immunoreactivity.

P2Y2 receptors and water transport in the kidney

The kidneys play a critical role in the maintenance of water homeostasis. This is achieved by the inherent architecture of the nephron along with the expression of various membrane transporters and channels that are responsible for the vectorial transport of salt and water. The collecting duct has become a focus of attention by virtue of its ability to transport water independent of solutes (free-water transport), and its apparent involvement in various water balance disorders. It was originally believed that the water transport capability of the collecting duct was solely under the influence of the circulating hormone, arginine vasopressin (AVP). However, during the past decade, locally produced autocrine and/or paracrine factors have emerged as potent modulators of transport of water by the collecting duct. Recently, much attention has been focused on the purinergic regulation of renal water transport. This review focuses on the role of the P2Y2 receptor, the predominant purinergic receptor expressed in the collecting duct, in the modulation of water transport in physiological and pathophysiological conditions, and its therapeutic potential as a drug target to treat water balance disorders in the clinic. Studies carried out by us and other investigators are unravelling potent interactions among AVP, prostanoid and purinergic systems in the medullary collecting duct, and the perturbations of these interactions in water balance disorders such as acquired nephrogenic diabetes insipidus. Future studies should address the potential therapeutic benefits of modulators of P2Y2 receptor signalling in water balance disorders, which are extremely prevalent in hospitalised patients irrespective of the underlying pathology.