A novel nitric oxide scavenger in combination with cyclosporine A ameliorates experimental autoimmune encephalomyelitis progression in mice

Journal of Neuroimmunology 138 (2003) 56 – 64 www.elsevier.com/locate/jneuroim

A novel nitric oxide scavenger in combination with cyclosporine A ameliorates experimental autoimmune encephalomyelitis progression in mice Corinne G. Jolivalt a, Randy B. Howard a, Long S. Chen a, Andrew P. Mizisin b, Ching-San Lai a,* b

a Medinox Inc., Suite 201, 11575 Sorrento Valley Road, San Diego, CA 92121, USA Department of Pathology, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

Received 13 December 2002; received in revised form 7 March 2003; accepted 7 March 2003

Abstract Immunotherapy improves experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), while excessive production of nitric oxide (NO) has been implicated in the pathogenesis of this disease. Here, we show that disease progression in SJL/J mice with EAE is improved after treatment with either a subtherapeutic dose of cyclosporine A (CsA) or NOX-100, a nitric oxide scavenger. Importantly, the impact of subtherapeutic doses of CsA in combination with NOX-100 on disease progression in EAE was greater than that attained with either agent alone and led to near total protection. CNS inflammation and gene expression of proinflammatory cytokines and iNOS were also significantly reduced after treatment. These observations point to the potential therapeutic utility of NOX-100 as a dosereducing agent for CsA in the treatment of MS. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Experimental autoimmune encephalomyelitis; Nitric oxide; NO scavenger; Cyclosporine A; Cytokines

1. Introduction Multiple sclerosis (MS) is a chronic relapsing autoimmune disease of the central nervous system (CNS). While the complex pathogenesis of MS is not completely understood, it includes inflammation, demyelination, and consequent destruction of oligodendrocytes and neurons (Raine, 1994; Trapp et al., 1998; Noseworthy et al., 2000). Inflammation is generally believed to involve aberrant T-cell reactivity to myelin antigens, resulting in demyelination characteristic of the initial relapsing – remitting phase of MS (Miller and Karpus, 1994) that precedes progressive irreversible neurological disability (Weinshenker et al., 1989). Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease that, in various rodents and other species, models acute and chronic aspects of MS, including demyelination (Arnason, 1983; Zamvil and Steinman, 1990). As an

* Corresponding author. Tel.: +1-858-793-4820; fax: +1-858-7934823. E-mail address: [email protected] (C.-S. Lai).

experimental animal model, EAE has been and continues to be used to investigate the pathogenesis of MS, and also to assess the efficacy of potential therapeutic agents for this debilitating disease. The immunosuppressive drug, cyclosporine A (CsA), has been shown to have a protective effect on rats, guinea pigs, and monkeys with EAE (Bolton et al., 1982a) and on humans with MS (Rudge et al., 1989). CsA suppresses cell-mediated immune reactions, such as allograft rejection (Calne et al., 1981; for review, see Hong and Kahan, 2000), EAE (Borel et al., 1976; Bolton et al., 1982b), and Freund’s adjuvant arthritis (Hambleton and McMahon, 1990; Boissier et al., 1992). CsA specifically and reversibly inhibits immunocompetent lymphocyte proliferation, particularly T cells (Klaus and Kunkl, 1983; Palacios and Moller, 1981), and inhibits interleukin-2 (Il-2) release and lymphokine production. However, CsA causes renal and hepatic toxicity and, consequently, cannot be used for long-term treatment of MS. To date, CsA has had little or no effect on the course of MS in human clinical trials (Kappos, 1988; Rudge et al., 1989; The Multiple Sclerosis Study Group, 1990). The proinflammatory cytokines, tumor necrosis factor (TNF) a and interferon (IFN) g, have been implicated in

0165-5728/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(03)00097-3

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2. Materials and methods 2.1. Material Eight-week-old female SJL/J mice (Jackson Laboratory, Bar Harbor, ME) were used in this study. Animals were housed three to six per cage, and provided with food and water ad libitum. NOX-100, a dithiocarbamate derivative, is a low-molecular-weight, water-soluble, NO-scavenging compound (Medinox, San Diego, CA). NOX-100 scavenges excess of NO by formation of a complex with iron without affecting physiological levels, as shown in vivo using EPR (electronic paramagnetic resonance) spectrometry (Lai and Komarov, 1994, 1995). CsA (Sandimmune) was obtained from Sandoz (Basel, Switzerland). Protocols for this study were reviewed and approved by IACUC (Institutional Animal Care and Use Committee) at Medinox. 2.2. Disease induction and clinical evaluation Fig. 1. Amelioration of the clinical course of EAE by NOX-100, CsA, or NOX-100 in combination with CsA up to 24 days after induction of EAE. (A) Mean individual daily clinical score between days 9 and 24 after induction of EAE. Each point represents the mean individual daily score of eight mice per group. (B) Cumulative scores between days 9 and 24. Each bar represents the mean F S.E.M. for eight mice. *p < 0.05 versus olive oil group. ##p < 0.01 versus CsA group.

the pathogenesis of MS (Issazadeh et al., 1995; Merrill and Benveniste, 1996; Navikas and Link, 1996; Taupin et al., 1997). TNFa increases NFnB expression, which may then increase transcription of inducible nitric oxide synthase (iNOS) mRNA (Kwon and George, 1999). Induction of iNOS leads to excess production of NO and consequent cytotoxic effects, which may be mediated in part by the formation of peroxynitrite when NO reacts with superoxide (van der Veen et al., 1997). The beneficial effect of iNOS inhibitors, such as aminoguanidine (Cross et al., 1994) and Lovastatin (Stanislaus et al., 1999), which block induction of iNOS, TNFa, Il-1h, and Il-6 in cultured rat astrocytes, microglia, and macrophages (Pahan et al., 1997) point to a potential role for NO in the pathogenesis of EAE (for a review, see Willenborg et al., 1999) and possibly MS. In this study, we investigated the efficacy of NOX-100, a NO scavenger and novel antiinflammatory agent, at preventing disease progression in EAE. NOX-100 is a dithiocarbamate capable of scavenging excess NO when complexed to iron and, as shown for other dithiocarbamates (Martinez-Martinez et al., 1997), acts as an immunosuppressant. Additionally, the impact of combination therapy using both NOX-100 and CsA was assessed in order to investigate the potential of NOX-100 at lowering the effective dose of CsA needed to prevent disease progression in EAE.

On day 0, acute EAE was induced by injecting into each hind foot pad an emulsion consisting of Complete Freud’s Adjuvant (CFA, Difco, Detroit, MI), Mycobacterium tuberculosis H37 RA (4 mg/ml, Difco), and myelin basic protein (MBP) proteolipid peptide 139 – 151 (H-HSLGKWLGHPDKF-Amide, Tuohy et al., 1989) (0.4 mg/ml, Research Genetics, Huntsville, AL). Three hundred nanograms of Pertussis toxin (List Biological Laboratories, Campbell, CA) in phosphate-buffered saline (PBS) was injected intraperitoneally 1 day after inoculation. Nine days after inoculation, mice were scored every day according to the

Fig. 2. Daily incidence of EAE after treatment with NOX-100, CsA, or NOX-100 in combination with CsA. Mean daily incidence up to day 16 after induction of EAE for all five experiments.

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following scale: 0—normal, 1—tail drop, 2—tail drop and hind leg weakness (mouse can still walk), 3—tail drop and hind leg paralysis (mouse can crawl), 4—total paralysis, and 5—death. 2.3. Treatment A total of 160 mice were used in five separate experiments. Each experiment included four treatment groups of eight mice each. As a vehicle control for mice receiving CsA, a group of mice were injected intraperitoneally daily with 2.5 ml/kg of olive oil (Sigma). The remaining mice were either treated with CsA alone, NOX-100 alone or both CsA and NOX-100. CsA (2 mg/ml) was prepared in olive oil containing 2% ethanol and injected intraperitoneally at 2.5 ml/kg body weight. NOX-100 was dissolved in drinking water (5 mg/ml) that was changed daily. Vehicle control and CsA treatment were initiated on day 5 after EAE induction and continued daily until the end of each experiment. NOX100 treatment began 4 days after EAE induction and continued until the end of each experiment. 2.4. Histology Sixteen days after induction in EAE in two different experiments, animals were sacrificed by an overdose of isoflurane before removing vertebral columns and fixing in 10% buffered formalin (Fischer, Pittsburgh, PA). After 24– 48 h, spinal cords were dissected out and kept in 10% formalin until processing. Tissues were embedded in paraffin, sectioned at 5 Am on a Reichert microtone, and stained with hematoxylin – eosin. For each animal, the number of infiltrating cells as well as the number of vessels surrounded by infiltrating cells were counted in six representative fields using a Jeneval 250-CF light microscope fitted with a camera lucida attachment. The average number of infiltrating cells as well as an average number of vessels surrounded by infiltrating cells in the six fields was calculated for each animal and then used to calculate group averages.

Fig. 3. Spinal cord from mice with untreated EAE or EAE treated with CsA, NOX-100, or the combination of CsA and NOX-100. Representative sections from EAE mice receiving olive oil (A), CsA alone (B), NOX-100 alone (C), or the combination of CsA and NOX-100 (D). In (A), an intense inflammatory infiltrate is evident beneath the pia mater and surrounding the longitudinal blood vessel profile. Less intense infiltrates were apparent in EAE mice receiving CsA alone (B) or NOX-100 alone (C). The combination of both agents markedly attenuated infiltrates (D) such that in some instances, the spinal cords were indistinguishable from those of normal SJL/J mice (data not shown). Hematoxylin and eosin. Scale bar = 40 Am and applies to all micrographs.

2.5. mRNA analysis On day 16, mice were sacrificed by an overdose of isoflurane. Brain and spinal cord were harvested and quick-frozen in liquid nitrogen. RNA was isolated using a

total RNA isolation kit (Promega, Madison, WI) for brain and using Trizol (Life Technologies, US) for spinal cord. RNA were then subjected to a Multiprobe RNAse Protection Assay following the manufacturer’s protocol (Pharmingen,

Table 1 Effect of NOX-100, CsA, or NOX-100 in combination with CsA on the cumulative clinical score of EAE mice

Olive oil CsA NOX-100 CsA + NOX-100

Experiment 1

Experiment 2

Experiment 3

Experiment 4

Experiment 5

6.8 F 0.88 4.0 F 1.24 5.9 F 1.27 0F0

10.68 F 1.34 6.0 F 2.79 2.88 F 1.29 1.67 F 0.2

7.7 F 2.85 7.8 F 3.24 5.5 F 1.23 0F0

11.0 F 0.51 4.66 F 1.35 4.16 F 1.66 1 F 0.63

10.5 F 1.36 2.66 F 1.08 5.33 F 1.07 1.66 F 1.08

Compilation of data obtained in five different experiments until day 16.

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San Diego, CA) using a template for iNOS, TNFh, TNFa, Lymphotoxin (LT) h, Il-6, IFN g, IFN h, tumor growth factor (TGF) h1, TGFh2, L32, and glyceraldehydes-3-phosphate deshydrogenase (GAPDH). The protected fragments were separated by electrophoresis and signals were analyzed by autoradiography. 2.6. Nitrate levels Nitrate levels were assessed in serum on the day of sacrifice. Blood was collected by cardiac puncture into a serum tube (Microtainer) under isoflurane anesthesia. Serum (100 Al) was treated with 200 Al of acetonitrile and then centrifuged 10 min at 14,000g and 4 jC. The supernatant was analyzed by high-performance liquid chromatography (HPLC) using an anion exchange column with a mobile phase of 20 mM NaOH. 2.7. Statistical analysis Unless otherwise noted, data are presented as means F S.E.M. Differences between group means were assessed with an unpaired, two-tailed t-test.

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3. Results 3.1. Clinical disease The clinical course of EAE in a representative experiment conducted for 24 days after induction of EAE is shown in Fig. 1. The untreated EAE mice receiving only olive oil developed hind leg weakness or paralysis, with the first symptoms appearing between 9 and 10 days after inoculation and reaching a peak score within 3 –5 days later. Disease progression in mice treated with CsA had a similar evolution; however, there was a faster and greater partial recovery than in vehicle-treated control mice (Fig. 1A). Treatment with NOX-100 alone attenuated disease severity, but the course and the severity of EAE was significantly reduced by administering both CsA and NOX-100 in comparison to untreated ( p < 0.05) and CsA-treated ( p < 0.01) mice (Fig. 1A and B). For this particular study, only one out of eight mice receiving both CsA and NOX-100 showed signs of EAE (e.g. a weak tail at day 17). One mouse in the vehicletreated control group and one in the CsA-treated group died from EAE, which explains the larger SEM of cumulative scores for these two groups (Fig. 1B).

Fig. 4. Morphometric assessment of inflammatory infiltrates in spinal cord of EAE mice. Treatment with CsA, NOX-100, or the combination of CsA and NOX100 reduced the degree of cell infiltration (A) and the number of vessels surrounded by infiltrating cells (B). Each bar represents the mean F S.E.M. for eight mice. *p < 0.05, **p < 0.01 versus olive oil group. #p < 0.05, ##p < 0.01 versus normal SJL mice.

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Fig. 5. Effects of treatment with NOX-100, CsA, or CsA and NOX-100 on the mRNA expression of various cytokines in the brain and spinal cord of mice with EAE. The intensity of the mRNA expression of each cytokine was normalized to that of the L32 housekeeping gene. Each bar represents the mean F S.E.M. for eight mice. *p < 0.05 versus olive oil group. Normal: normal mice without EAE.

Daily incidences are shown in Fig. 2 as means of all five experiments, and 16-day cumulative clinical scores are given in Table 1 for all five experiments. As described for the 24-day experiments (Fig. 1), the combination of CsA and NOX-100 substantially reduced disease incidence (Fig. 2) and cumulative clinical scores (Table 1). 3.2. Histology Infiltrating cells (i.e. monocytes and polymorphonuclear cells) were significantly more numerous in the spinal cord sections of the vehicle control mice than in those from normal SJL/J mice (Figs. 3 and 4A). Indeed, spinal cords from normal SJL mice without EAE had only few scattered inflammatory cells and did not show any vessels surrounded by infiltrating cells (data not shown). Histological lesions seen in the treated EAE groups were significantly reduced after all treatments, with the combination of CsA and NOX-100 being the most effective (Figs. 3 and 4). Indeed, the combination treatment reduced infiltration to a level close to normal mice and almost no vessels were surrounded by infiltrates. In addition, on spinal cord sections stained for MBP, demyelination visualized by a lack of staining was occasionally observed for vehicletreated mice but not in the treatment groups (data not shown).

3.3. Cytokine expression In the brain and spinal cord, TNFa mRNA was significantly induced in vehicle control animals, which developed severe EAE, compared to normal SJL/J mice ( p < 0.05) (Fig. 5). Treatment with NOX-100 or CsA significantly reduced TNFa mRNA expression in brain ( p < 0.05), while the combination of NOX-100 and CsA significantly reduced TNFa mRNA expression in both brain and spinal cord ( p < 0.05). As with TNFa, mRNA expression for Il-1h, Il-

Fig. 6. iNOS mRNA expression in the CNS of EAE mice. The intensity of the mRNA expression of iNOS was normalized to that of the L32 housekeeping gene. Each bar represents the mean F S.E.M. for eight mice. Normal: normal mice without EAE.

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Fig. 7. Comparison of the serum nitrate level of EAE mice. Treatment with NOX-100 or NOX-100 and CsA significantly reduced the serum nitrate level in EAE mice. Each bar represents the mean F S.E.M. for eight mice. *p < 0.05, **p < 0.01 versus olive oil group. SJL normal: normal SJL/J mice without EAE.

6, and IFNg was significantly increased in both brain and spinal cord of mice treated with vehicle compared to normal SJL/J mice ( p < 0.05) (Fig. 5). In all cases, mRNA expression was higher in spinal cord than brain. Cytokine mRNA expression was reduced in all three treatment groups, with a trend towards a further reduction with the combination of CsA and NOX-100. 3.4. iNOS expression iNOS expression was significantly induced by EAE in both brain and spinal cord (Fig. 6). Treatment with CsA or NOX-100 attenuated iNOS mRNA expression in both brain and spinal cord. The attenuation of iNOS expression was even more pronounced when mice were treated with the combination of CsA and NOX-100. The pattern of iNOS expression with respect to the effect of treatment was similar to that observed for cytokines (Fig. 5). 3.5. Nitrate levels Nitrate levels, reflecting NO levels, were significantly increased in the plasma of EAE mice treated with vehicle compared to normal mice ( p < 0.01) (Fig. 7), an increase consistent with the increase in iNOS expression after EAE induction (Fig. 6). Compared to vehicle-treated EAE mice, treatment with NOX-100 or CsA combined with NOX-100 significantly ( p < 0.01 and 0.05, respectively) reduced plasma nitrate levels to those seen in normal mice (Fig. 7), consistent with NOX-100’s ability to scavenge excess NO without affecting physiological levels.

4. Discussion The present study tested the efficacy of CsA and/or NOX-100 in an animal model of EAE in an attempt to reduce the effective dose of CsA and side effects associated with this immunosuppressive agent. Bolton et al. (1982a) showed that CsA at a dose of 50 mg/kg suppressed EAE in

rats. Later work demonstrated that lower concentrations of CsA had no effect (Polman et al., 1988; Pender et al., 1990) or, when used at a dose of 8 mg/kg, delayed the disease but increased the frequency of second and third episodes (McCombe et al., 1999; Harness et al., 2001). Recently, treatment with a combination of CsA and NOX-100 prolonged cardiac allograft survival, by reducing inflammation, plasma nitrate, and myocardial nitrosyl (Pieper et al., 2000). Here, a subtherapeutic dose of CsA (5 mg/kg) in combination with NOX-100 significantly delayed disease progression and reduced its severity in an EAE mouse model, as well as in EAE rats (data not shown). Our data also demonstrate that NOX-100, when administered alone, effectively reduced the severity of EAE. EAE in mice is mainly an inflammatory disease, although demyelination is apparent but not predominant as seen in spinal cord sections stained for MBP in our experiments (data not shown). Our data demonstrate that the benefits of NOX-100 and CsA assessed by clinical scores correlate with histological observations as well as with mRNA expression of cytokines and iNOS in the CNS. Cytokines play an important role in EAE. Interleukins (especially Il-4, Il-5, Il-6, and Il-13) stimulate B cell production of antibodies (Thomson, 1994), while TNFa, LTh, and IFNg mediate myelin damage directly and/or indirectly via activation of macrophages that produce proteases, TNFa, NO, and H2O2 (Navikas and Link, 1996). In our study, mRNA expression of IFNg, LTh, Il-6, TNFa were significantly increased in brain and spinal cord of vehicle-treated EAE mice, consistent with the proinflammatory cytokine profile in MS (Rieckmann et al., 1995; Navikas and Link, 1996). Elevated TNFa expression in EAE was significantly reduced by all three treatments but to a greater extent when CsA and NOX-100 were used together. A similar pattern was observed for the other cytokines examined as well as for iNOS expression. Interestingly, reductions in IFNg and Il-2 mRNA expression have been reported in a rat model of EAE after treatment with CsA (Harness et al., 2001). The effect of CsA on cytokine expression in the brain and spinal cord was expected, as CsA is known to be an antiinflammatory agent.

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The reduction of cytokine expression in EAE mice after treatment with NOX-100 points to the antiinflammatory activity of this agent, consistent with similar effects of other dithiocarbamates (Martinez-Martinez et al., 1997). In fact, dithiocarbamates have been shown to block NFnB activation in cell culture (Schreck et al., 1992), prevent NFnB activation and the resulting inflammation in carrageenaninduced pleurisy and collagen-induced arthritis (Cuzzocrea et al., 2002), and inhibit the synthesis of Il-6 and Il-8 (Mun˜oz et al., 1996). In a recent study, the combination of NOX-100 and CsA inhibited NFnB, normalized NO levels and prolonged cardiac allograft survival (Roza et al., 2000). Inhibition of NFnB activation by dehydroepiandrosterone (DHEA) treatment has been shown to decrease expression of IFNg, Il-12, TNFa, and NO in EAE (Du et al., 2001). An additional effect of inhibiting cytokine expression may be the reduction of iNOS expression in brain and spinal cord from mice treated with NOX-100, CsA or both. TNFa, Il-1h, and IFNg can induce iNOS expression via NFnB (Xie et al., 1994; Martin et al., 1994; Taylor et al., 1998; MarksKonczalik et al., 1998; Kwon and George, 1999). NOX-100 treatment led to decreased expression of TNFa and IL-1h after hemorrhage (Menezes et al., 1999) and, similarly, treatment with NOX-100 reduced TNFa and Il-6 expression in rat liver after endotoxin challenge (Nadler et al., 2001). Therefore, treatment with NOX-100, CsA or both may reduce cytokine expression, leading to a decrease in iNOS induction and, therefore, decreased NO production. NO is increased in murine CNS during EAE (Lin et al., 1993) and iNOS is upregulated in experimentally induced neurologic diseases (Koprowski et al., 1993). Taken together these data suggest that the combination of CsA and NOX-100 may have synergistic antiinflammatory effects. NO produced by macrophages (Murphy et al., 1990) at the site of inflammation could alter myelin proteins by oxidation via peroxynitrite formation. Nitrotyrosine immunoreactivity, a marker of peroxynitrite formation, is prevalent in CNS tissues in animal models of MS and in humans with this disease (Cross et al., 1997, 1998; van der Veen et al., 1997). Recently, a role for peroxynitrite in the pathogenesis of EAE, distinct from that of NO, has been proposed (Cross et al., 2000). Reductions in hepatic peroxynitrite formation in hemorrhaged rats have been attributed to the NO-scavenging property of NOX-100 (Menezes et al., 1999). Here, reduction in iNOS expression by combined NOX-100 and CsA treatment resulted in a concomitant decrease in free NO as assessed by plasma nitrate levels, which conceivably could decrease peroxynitrite formation. Thus, in addition to antiinflammatory properties of NOX100, its NO-scavenging ability may help protect myelin by reducing lipid peroxidation and subsequent degradation. There is increasing acceptance that NO may have a protective as well as destructive role in EAE (for a review, see Willenborg et al., 1999). NO can modulate immune expression in a number of ways that could ameliorate EAE. By inhibiting macrophage Ia expression (Sicher et al.,

1994), NO could prevent T-cell expansion due to a lack of macrophage antigen presentation. NO also has a direct inhibitory effect on T-cell proliferation (Albina and Henry, 1991), which appears specific to the encephalitogenic Th1 CD4+ population (Taylor-Robinson et al., 1994). NO also downregulates expression of selectins, and cell adhesion molecules that mediate lymphocyte migration (Kubes et al., 1991) can induce apoptosis and necrosis of these cells (Okuda et al., 1997) and protect oligodendrocytes from lipid peroxidation-induced injury (van der Veen and Roberts, 1999). The exacerbated development of EAE in iNOS knock-out mice (Fenyk-Melody et al., 1998; Sahrbacher et al., 1998) further points to a protective role for NO in this experimental disease. In our study, the severity of EAE correlated with the increase in iNOS expression and NO levels, and treatment with NOX-100 alone or the combination of NOX-100 and CsA reduced iNOS expression and normalized NO levels while ameliorating clinical signs and infiltrates in spinal cord. It is important to note that here treatment with NOX-100 alone or in combination with CsA did not eliminate iNOS expression or NO, but rather reduced these parameters to levels present in normal animals, levels that may be sufficient to promote the protective effects of NO suggested above. In summary, the impact of subtherapeutic doses of CsA in combination with NOX-100 on disease progression in EAE was greater than that attained with either agent alone, and led to near total protection. CNS inflammation and gene expression of proinflammatory cytokines and iNOS were also significantly reduced after treatment with these agents. The greater effect of the combination therapy may result from synergistic antiinflammatory properties of each agent, plus the NO-scavenging property of NOX-100. Furthermore, these data indicate a potential therapeutic value of a NO scavenger in preventing development of MS. Further investigation is needed to confirm these possibilities.

Acknowledgements The authors would like to thank Dr. H. Powell for helpful discussions, and B. Garrett and R. Ticsay for their technical assistance.

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