Therapeutic Effect of EDTA in Experimental Model of Multiple Sclerosis

Introduction

Multiple sclerosis (MS) is a chronic, immune-mediated demyelinating disease of the central nervous system (CNS) with unknown etiology. It affects approximately 2 million people worldwide [1, 2]. The pathogenesis of the disease is characterized by the activation and infiltration of mononuclear cells, predominantly antigen-specific CD4+ and CD8+ T cells and B cells, in the central nerv-ous system, reactivation by resident antigen presenting microglial cells, secretion of proinflammatory cytokines/chemokines along with generation of other inflamma-tory mediators, such as complement components, highly reactive free radicals such as reactive oxygen species

(ROS) and reactive nitrogen species (RNS) resulting in the demyelination of axons [3].

Experimental autoimmune encephalomyelitis (EAE) is currently the most commonly used animal model for the study of MS. This model causes inflammation and demyelination similar to the disease manifestation seen in humans [1]. EAE is a T cell-mediated autoimmune disease of the CNS, in which the myelin oligodendrocyte glycoprotein (MOG) is an autoantigen recognized by autoreactive T cells. Migration of encephalitogenic T cells to the CNS plays an essential role in the development of EAE [4]. The Th1 effector cell subset responsible for production of pro-inflammatory cytokines such as IFN-γ has traditionally been implicated in the pathogenesis

Immunopharmacology and ImmunotoxicologyImmunopharmacology and Immunotoxicology, 2010; 32(2): 321–326

2010

32

2

321

326

Address for Correspondence: Abbas Mirshafiey, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran-14155, Box: 6446, Iran. Fax: +98-21-66462267. E-Mail: [email protected]

23 June 2009

14 September 2009

15 September 2009

0892-3973

1532-2513

© 2010 Informa UK Ltd

10.3109/08923970903338367

O R I G I N A L A R T I C L E

Therapeutic Effect of EDTA in Experimental Model of Multiple Sclerosis

G. Mosayebi1, D. Haghmorad2, S. Namaki2, A. Ghazavi1, P. Ekhtiari2, and Abbas Mirshafiey2

1Department of Immunology, Arak University of Medical Sciences, Arak, Iran, and 2Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

AbstractMultiple sclerosis (MS) is a chronic, neurodegenerative disease that causes central nervous system (CNS) demyelination and affects approximately 2 million people worldwide. The aim of the present study was to test the therapeutic effect of ethylene diamine tetra acetic acid (EDTA) in an experimental model of MS. The experiment was done on male C57BL/6 mice aged 6–8 weeks. The experimental autoimmune encephalo-myelitis (EAE) was induced using 250 μg of the MOG35-55 peptide emulsified in CFA and injected subcuta-neously on day 0 over two flank areas. In addition, 250 ng of pertussis toxin in 400 μl PBS was injected i.p. on days 0 and 2. In the treatment group, EDTA was administered i.p. at a dose 75 mg/kg/day. The mice were sacrificed 21 days after EAE induction and blood samples were taken from their hearts. The brains of mice were removed for histological analysis and their isolated splenocytes were cultured. Results: Our results showed that treatment with EDTA caused a significant delay in the time of onset and a significant reduction in severity of the EAE. Histological analysis indicated that there was not any plaque in the group of EDTA-treated mice whereas 5 ± 1 plaques were detected in controls. The density of mononuclear infiltration in the CNS of EDTA-treated mice was lower than controls. In the group of EDTA-treated mice, using the FRAP test, total serum antioxidant power was high and significant in comparison to the controls. Moreover, the serum level of nitric oxide in the treatment group was significantly less than the control group, and, also, the levels of IFN-γ in the cell culture supernatant of treated mice splenocytes was lower than control group. These data indicate that EDTA therapy can effectively attenuate EAE progression.

Keywords: MS; EAE; EDTA; multiple sclerosis; chelation therapy the

IPI

434014

(Received 23 June 2009; revised 14 September 2009; accepted 15 September 2009)

ISSN 0892-3973 print/ISSN 1532-2513 online © 2010 Informa UK LtdDOI: 10.3109/08923970903338367 http://www.informahealthcare.com/ipi

322 G. Mosayebi et al.

of certain autoimmune diseases including EAE [5].The EAE is thought to be a T cell-mediated autoimmune disease, so that, both Th1 and Th17 cells were thought to be responsible for the inflammatory demyelination in multiple sclerosis and EAE. Th2 and Foxp3 regulatory T cells (Treg) and related cytokines such as IL-5 and IL-10 have been shown to be important in the resolution stages of the disease [6].

Studies of brain tissue, serum and cerebrospinal fluid (CSF) from patients with MS have consistently found an upregulation of several matrix metalloproteinases (MMPs) in MS. In particular, MMP-9 has been shown to be altered in MS in several studies [7].

Oxidative stress contributes to the pathogenesis of a wide number of diseases including rheumatoid arthritis, cardiovascular diseases, and neurological disorders via the overproduction of ROS and RNS such as hydrogen peroxide (H2O2), superoxide and peroxynitrite [8]. Ethylene diamine tetra acetic acid (EDTA) has beneficial effects via their anti-oxidant abilities to detoxify H2O2 and O2 [8].

EDTA and its salts are substituted diamines; these ingredients function as chelating agents in cosmetic for-mulations by combining with polyvalent metal cations in solution to form soluble ring structures [9].

Chelation therapy can be defined as the use of repeated intravenous administration of EDTA, as a treatment for a variety of diseases [10]. For many years, EDTA has been approved by the Food and Drug Administration (FDA) for use in the treatment of some types of heavy metal poi-soning [11], and it has been touted as a safe alternative treatment for atherosclerotic vascular disease [12].

The aim of this study was to determine the therapeutic effect of EDTA on the improvement of EAE induced by MOG 35-55 in mice and to elucidate the possible mecha-nism involved.

Material and Methods

Mice

C57BL/6 male mice, 6–8 weeks of age, were obtained from the Pasteur Institute of Iran. Mice were randomly separated into three groups: normal, control and treat-ment with 8 mice in each group. All mice were housed in groups of four in cages on a 12-hour light-dark cycle and with free access to food and water. All procedures involving animals were performed according to the of the animal ethics guidelines approved by Tehran University of Medical Science.

EAE induction

Initially, anesthesia was done by ether and each mouse then received a subcutaneous injection of 250 μg synthetic

MOG35-55 (MEVGWYRSPFSRVVHLYRNGK; Diapharm Ltd., Moscow, Russia) in 100 μl PBS mixed with 100 μl of complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA). A 100 μl volume was injected subcutanously over two flank areas. Mice were also intraperitoneally (i.p.) injected with 250 ng of pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) in 400 μl of PBS. A second, identical injection of pertussis toxin was given after 48 h [1, 6].

EDTA (Merck, Munchen, Germany) in 100 μl PBS was administered i.p. at dose 75 mg/kg/day in the treatment group [13, 14]. The control group (after EAE induction) and normal group received 100 μl PBS. Daily treatment was carried out from day 4 to day 13 post-immunization. Clinical assessment was performed daily from day 9 post-immunization until day 21. Mice were monitored daily and assessed by clinical score.

The clinical grade was scored as follows: 0, no clinical sign; 1, partial loss of tail tonicity; 2, complete loss of tail tonicity; 3, flaccid tail and abnormal gait; 4, hind leg paralysis; 5, hind leg paralysis with hind body paresis; 6, hind and foreleg paralysis; 7, moribund or death [15].

Histology assessment

To evaluate histopathology, mice were sacrificed after 21 days following anaesthetization by perfusion ketamin and zylasin through the heart. Brains were dissected out and fixed with neutral 10% formalin overnight and routinely embedded in paraffin wax. Brain sections (8μm thick) were stained with hematoxylin and eosin (H&E), used to highlight areas of inflammation by darkly staining the nuclei of mononuclear cells. Immune cells stained with H&E were counted in a blinded manner under a light microscope using a 10x magnification with 12 sections examined per animal. Each section was counted manu-ally before being combined to give a total for the animal [16].

FRAP Test

Total antioxidant power was measured according to the FRAP test. In the FRAP test antioxidants in the sample reduce ferric tripyridyltriazine complex (Fe3+- TPTZ), to a blue colored ferrous form (Fe2+), with an increase in absorbance at 593 nm.

The working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 1 volume of 10 mmol/L TPTZ (2, 4, 6 Tris(pyridyl)-s -tri-azine) (Sigma-Aldrich, St. Louis, MO, USA) in 40 mmol/L HCl and with 1 volume of 20 mmol/L FeCl3 prepared in deionized water. The working FRAP reagent was then warmed to 37°C and its absorbance against water was read at 593 nm (reagent blank). Subsequently, 30 μl of serum sample was added to 970 μl of FRAP reagent and the absorbance was monitored within 4 min. The results

Therapeutic Effect of EDTA in Experimental Model of Multiple Sclerosis 323

were expressed as μmol of ferric antioxidant power (the FRAP value) and were compared with the standard curve prepared using FeSO4, 7H2O 1mM in a range of concen-tration from 125 to 1000 μM [17].

Nitrite assay

NO was assayed by measuring nitrite as the end product of reaction, which was determined by a colorimeter assay based on the Griess reaction. Griess reagent was prepared by dissolving 1g sulfanilamide in 100 ml phosphoric acid 5% mixed with 0.1g naphtyl ethylene diamine-HCl (NED) in 100 ml distilled water. Serum sample (100 μl) was mixed with 100 μl of Griess reagent at room temperature for 10 min. Absorbance was measured using a microplate reader at 550 nm. The concentration of nitrite was deter-mined by the standard curve of sodium nitrite 0.1 molar prepared in distilled water [18, 19]

IFN-γ evaluation

Single cell suspensions were obtained from spleens 21 days post-immunization. Splenocytes isolated from mice were treated with lysis buffer to remove red blood cells. Cells were resuspended in PBS and a cell count was per-formed. 2 × 106 cells per well were cultured in 24-well cul-ture plates (Greiner Bio-One, Germany) in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, 50 μg/ml gentamicin-penicillin (Sigma), in the presence of 10 μg/ml MOG 35-55 peptide. Cultures were incubated for 72 hours in 5% CO

2 at 37°C. To assay cytokine, cells culture

supernatants were collected after stimulation with antigen. IFN- γ and IL-10 have been quantified by two-site sand-wich ELISA using a commercially available kit (Bender Med. Systems, Vienna, Austria) on cell culture superna-tants. Absorbance was read at 450 nm using microplate ELISA reader (Stat Fax 2100, Awarness, Phoenix, Arizona, USA). The concentration of cytokine was estimated from a standard curve generated with each run.

Statistics

Statistical analyses were performed by Mann–Whitney non-parametric test and the Student’s t-test for unpaired data. Results are reported as mean ± SD; P values <0.05 were considered to be statistically significant.

Results

Clinical findings

All immunized mice with MOG35-55 developed EAE. The time of onset, clinical course and severity of the disease differed consistently between treatment and control

groups (Figure 1). The disease was started earlier in the patient: the mean time of onset was 12 ± 1 and 10 ± 1 days after the first immunization, in treatment and control mice, respectively. The mean severity score of the acute disease was almost two times higher in control mice (4.87 ± 0.83) than in treated mice (2.6 ± 1.92) (p < 0.05). These effects were led to significant clinical improve-ment and delayed disease progression during 21 days of observation, indicating that EDTA can inhibit both development and progression of EAE.

Histology findings

We examined whether it could be a correlation between the clinical symptoms of EAE with histopathology of CNS in control and treated mice. Histology analysis indicated that there was no plaque in the group of EDTA-treated mice whereas 5 ± 1 plaques (in each section) were detected in the control group as shown in Figure 2B. The density of mononuclear infiltration in the CNS of

Mean clinical score Comparison

0

1

2

3

4

5

6

1 3 5 7 9 11 13 15 17 19

Days post immunisation

Mea

n cl

inic

al s

core

EDTAControl

*

Figure. 1. Mean clinical scores of EAE induced by MOG 35-55 in con-trol mice and in mice treated with EDTA. All mice were immunized with 250μg MOG 35-55 emulsified in CFA and screened every other day for the presence of clinical signs of EAE. *p < 0.05

C

DB

A

Figure 2. Brain sections from normal (A), control (B) and treated mice (C, D) were stained with H&E. Leukocyte infiltration are less evident in the EDTA treated mice in comparison with control mice. In Figufre 2B, plaque is shown with arrow indicating a high infiltration of mononu-clear cells, whereas in the treatment group, there is no plaque.

324 G. Mosayebi et al.

EDTA-treated mice was lower than non-treated mice. As shown in Figure 2 the mice treated with EDTA displayed less inflammation in the CNS than did the control mice. These results indicate that the histopathology of CNS correlates with the clinical severity of EAE in treated and control mice.

FRAP evaluation

All mice were sacrificed 21 days after EAE induction and blood samples were taken from their hearts. FRAP reaction were performed on serum sample. Antioxidant capacity was expressed as the FRAP value. The results are shown in Figure 3. Treated mice showed high FRAP values (573.8 ± 148.69 μmol) compared to control mice (327.73 ± 11.24 μmol). Statistical analysis displayed that antioxidant activity in the treatment group is significantly higher than in the control mice (p < 0.001).

NO production findings

Griess reaction was performed on serum sample. As shown in Figure 4, NO production was significantly reduced in treated mice (5.21 ± 3.09 μM, p < 0.01), com-pared to control mice (11.14 ± 2.8 μM). In the normal group NO production was 4.7 ± 1.25 μM.

IFN-γ assessment

Splenocytes were obtained from mice and cultured for 72 h in the presence of MOG 35-55. The treatment group significantly exhibited lower levels of IFN-γ production in comparison to the patient group (Figure 5, 40 ± 25 ρg/ml and 1492 ± 443 ρg/ml, respectively, p < 0.001).

Discussion

EAE as experimental model of MS is an autoimmune dis-ease of CNS mediated by CD4+ T lymphocytes specific for autoantigens of the myelin sheath, including myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein peptide [20, 21]. Remitting/relapsing EAE (RR-EAE) induced by active immunization with the immunodominant epitope of MOG35-55 is characterized by an initial acute phase, followed by a series of remis-sions and relapses [22]. CD4+ Th1 cells and their proin-flammatory cytokines are suspected to be important in the pathogenesis of MS, and necessary for the induction of RR-EAE [23].

The cytokine profile and the nature of cellular infiltrate are markedly different during the clinical course of RR-EAE, so that the maximal expression of the proinflammatory cytokines IFN-γ and TNF-α are important mediators for disease induction. Th2

EDTA Ctrl Normal0

100

200

300

400

500

600

700

800

FRA

P µM

/Lit

EDTACtrlNormal

***

*

Figure 3. Antioxidant capacity was measured by the FRAP reaction. Data are presented as the mean ± S.D. of duplicate samples from 5 to 10 mice. *p < 0.05; ***p < 0.001.

EDTA Ctrl Normal0

2

4

6

8

10

12

14

16

Nitr

ite µ

M EDTACtrlNormal

** **

Figure 4. NO production was measured by the Griess reagent. Data are presented as the mean ± S.D. of duplicate samples from 5 to 10 mice. **p < 0.01.

EDTA Ctrl0

500

1000

1500

2000

2500

IFN

-γ p

g/m

l

EDTACtrl

***

Figure 5. Spleen cells were isolated from all mice on day 21 following immunization with MOG35-55 and cultured. The culture supernatants were collected after 72 h, and the levels of IFN-γ was measured by ELISA. The values are mean of triplicates, and the error bars represent SD. ***p < 0.001.

Therapeutic Effect of EDTA in Experimental Model of Multiple Sclerosis 325

cell clones specific for encephalitogenic peptides are unable to induce the disease and can inhibit Th1 autoimmune clones, presumably by secreting IL-4, IL-10, and TGF-ß [24].

Our findings suggest that EDTA is capable of sup-pressing a pre-activated immune system in the late effector phase leading into disease eruption. Although, this drug could penetrate into the CNS, but we could not test the time point related to the stage of disease progression, in which the encephalitogenic T-cells are initiating infiltration into the CNS and beginning to recruit macrophages and microglial cells. The dose-response experiments carried out in the present study demonstrating that, EDTA has an optimum therapeutic effect at dose 75mg/kg.

We assume that several mechanisms are involved in the therapeutic effect of EDTA. The removal of some divalent cations by EDTA as a matrix metalloproteinase (MMP) broad spectrum inhibitor may inhibit MMPs activity that involves in migration of leukocytes [25].

New data suggest a favorable antioxidant mechanism for EDTA, similar to that previously described for other chelating agents. In fact the EDTA complexes with metal ions such as Fe++ and Cu++ suppress superoxide and hydrogen peroxide activity [26]. In addition, Hininger et al. showed the beneficial antioxidant effects of EDTA chelating therapy [27]. Since oxidative stress contributes to the pathogenesis of many diseases, including MS and/or EAE and protection exerted by EDTA against CNS inflammation could be mediated by antioxidant property.

The pathogenic processes in EAE are mainly related to the secretion of IFN-γ [28, 29]. This study showed that treatment with EDTA can inhibit a pro-inflammatory Th1-based cytokine response and attenuate clinical signs in EAE. The results obtained in this research clearly show that EDTA therapy effectively reduces the severity of EAE-associated clinical symptoms and neuroinflam-mation in the brain. Moreover, EDTA therapy showed an appropriate cytokine response based on decreasing the proinflammatory Th1 cytokine, IFN-γ production. In addition, the results showed that the level of serum nitric oxide was significantly less than the control group, because the pathway over which IFN-γ induces NO production might be defeated through decreasing production of this cytokine [5].

NO inhibits T lymphocyte proliferation, preferentially of Th1 cells [30, 31]. Moreover, NO can function as a down-regulator of T cell migration, which might be one of the mechanisms by which NO exerts its protective effect in T cell-mediated autoimmune diseases [5]. Several studies have tried to explain the mechanisms of NO-mediated T cell migration inhibition: (1) NO provokes cytoskeletal changes in T lymphoblasts, and subsequent migration inhibition of T cells is mediated by a p70S6K-independent

mechanism [32]; (2) NO also inhibits T cell adhesion and migration by down-regulation of beta1-integrin expres-sion [33]; (3) NO can inhibit the expression of CD49d, the α4 subunit of VLA-4 that is critical for infiltration of inflammatory cells to the CNS and is a potential target for treatment of multiple sclerosis [5].

Conclusion

The results of this study suggest that EDTA modulates EAE, at least in part, by its antioxidant property, suppress-ing NO and IFN-γ production. This finding encourages further investigations of EDTA and in its role as a poten-tial candidate for developing more efficient therapeutic strategies against multiple sclerosis.

Declaration of interest: The authors report no con-flicts of interest. The authors alone are responsible for the content and writing of the paper.

References

1. Peiris M, Monteith GR, Roberts-Thomson SJ, Cabot PJ. A model of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice for the characterisation of intervention therapies. J Neurosci Methods 2007 Jul 30;163(2):245–54.

2. Furlan R, Kurne A, Bergami A, Brambilla E, Maucci R, Gasparini L, et al. A nitric oxide releasing derivative of flurbiprofen inhibits experimental autoimmune encephalomyelitis. J Neuroimmunol 2004 May;150(1–2):10–9.

3. Zaheer S, Wu Y, Bassett J, Yang B, Zaheer A. Glia maturation fac-tor regulation of STAT expression: a novel mechanism in experi-mental autoimmune encephalomyelitis. Neurochem Res 2007 Dec;32(12):2123–31.

4. Wang Y, Kai H, Chang F, Shibata K, Tahara-Hanaoka S, Honda S, et al. A critical role of LFA-1 in the development of Th17 cells and induction of experimental autoimmune encephalomyelytis. Biochem Biophys Res Commun 2007 Feb 23;353(4):857–62.

5. Xiao BG, Ma CG, Xu LY, Link H, Lu CZ. IL-12/IFN-gamma/NO axis plays critical role in development of Th1-mediated experi-mental autoimmune encephalomyelitis. Mol Immunol 2008 Feb;45(4):1191–6.

6. Jiang HR, Al Rasebi Z, Mensah-Brown E, Shahin A, Xu D, Goodyear CS, et al. Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis. J Immunol 2009 Jan 15;182(2):1167–73.

7. Yong VW, Zabad RK, Agrawal S, Goncalves Dasilva A, Metz LM. Elevation of matrix metalloproteinases (MMPs) in multiple scle-rosis and impact of immunomodulators. J Neurological Sciences 2007 Aug 15;259(1–2):79–84.

8. Fisher AE, Maxwell SC, Naughton DP. Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes. Biochem Biophys Res Commun 2004 Mar 26;316(1):48–51.

9. Lanigan RS, Yamarik TA. Final report on the safety assessment of EDTA, calcium disodium EDTA, diammonium EDTA, dipo-tassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA, trisodium EDTA, HEDTA, and trisodium HEDTA. Int J Toxicol 2002;21 Suppl 2:95–142.

10. Ernst E. Chelation therapy for coronary heart disease: an overview of all clinical investigations. Am Heart J 2000 Jul;140(1):139–41.

11. Lewin MR. Chelation therapy for cardiovascular disease. Review and commentary. Tex Heart Inst J 1997;24(2):81–9.

326 G. Mosayebi et al.

12. Seely DM, Wu P, Mills EJ. EDTA chelation therapy for cardio-vascular disease: a systematic review. BMC Cardiovasc Disord 2005;5:32.

13. Cory-Slechta DA, Weiss B. Efficacy of the chelating agent CaEDTA in reversing lead-induced changes in behavior. Neurotoxicology 1989 Winter;10(4):685–97.

14. Meldrum JB, Ko KW. Effects of calcium disodium EDTA and meso-2,3-dimercaptosuccinic acid on tissue concentra-tions of lead for use in treatment of calves with experimen-tally induced lead toxicosis. Am J Vet Res 2003 Jun;64(6): 672–6.

15. Skundric DS, Zakarian V, Dai R, Lisak RP, Tse HY, James J. Distinct immune regulation of the response to H-2b restricted epitope of MOG causes relapsing-remitting EAE in H-2b/s mice. J Neuroimmunol 2003 Mar;136(1–2):34–45.

16. Natarajan C, Muthian G, Barak Y, Evans RM, Bright JJ. Peroxisome proliferator-activated receptor-gamma-deficient hetero-zygous mice develop an exacerbated neural antigen-induced Th1 response and experimental allergic encephalomyelitis. J Immunol 2003 Dec 1;171(11):5743–50.

17. Nencini C, Cavallo F, Capasso A, Franchi GG, Giorgio G, Micheli L. Evaluation of antioxidative properties of Allium spe-cies growing wild in Italy. Phytother Res 2007 Sep;21(9):874–8.

18. Phizackerley PJ, Al-Dabbagh SA. The estimation of nitrate and nitrite in saliva and urine. Anal Biochem 1983 May;131(1):242–5.

19. Kayhan B, Aharoni R, Arnon R. Glatiramer acetate (Copaxone) regulates nitric oxide and related cytokine secretion in experi-mental autoimmune encephalomyelitis. ImmunolLetts 2003 Sep 8;88(3):185–92.

20. Tuohy VK, Lu Z, Sobel RA, Laursen RA, Lees MB. Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice. J Immunol 1989 Mar 1;142(5):1523–7.

21. Mendel I, Kerlero DR, Ben Nun A. A myelin oligodendrocyte glyc-oprotein peptide induces typical chronic experimental autoim-mune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells. Eur J Immunol 1995;25:1951–9.

22. McRae BL, Kennedy MK, Tan LJ, Dal Canto MC, Picha KS, Miller SD. Induction of active and adoptive relapsing experi-mental autoimmune encephalomyelitis (EAE) using an encepha-litogenic epitope of proteolipid protein. J Neuroimmunol 1992 Jun;38(3):229–40.

23. Beck J, Rondot P, Catinot L, Falcoff E, Kirchner H, Wietzerbin J. Increased production of interferon gamma and tumor necrosis

factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acta Neurologica Scand 1988 Oct;78(4):318–23.

24. Miller A, al-Sabbagh A, Santos LM, Das MP, Weiner HL. Epitopes of myelin basic protein that trigger TGF-beta release after oral tol-erization are distinct from encephalitogenic epitopes and medi-ate epitope-driven bystander suppression. J Immunol 1993 Dec 15;151(12):7307–15.

25. Sakakura Y, Hosokawa Y, Tsuruga E, Irie K, Yajima T. In situ localization of gelatinolytic activity during development and resorption of Meckel’s cartilage in mice. Eur J Oral Sciences 2007 Jun;115(3):212–23.

26. Foglieni C, Fulgenzi A, Ticozzi P, Pellegatta F, Sciorati C, Belloni D, et al. Protective effect of EDTA preadministration on renal ischemia. BMC Nephrol 2006;7:5.

27. Hininger I, Waters R, Osman M, Garrel C, Fernholz K, Roussel AM, et al. Acute prooxidant effects of vitamin C in EDTA chelation therapy and long-term antioxidant ben-efits of therapy. Free Radical Biol Med 2005 Jun 15;38(12): 1565–70.

28. Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner HL. Regulatory T cell clones induced by oral tolerance: suppres-sion of autoimmune encephalomyelitis. Science 1994 Aug 26;265(5176):1237–40.

29. Racke MK, Bonomo A, Scott DE, Cannella B, Levine A, Raine CS, et al. Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease. J Exper Med 1994 Nov 1;180(5):1961–6.

30. Allione A, Bernabei P, Bosticardo M, Ariotti S, Forni G, Novelli F. Nitric oxide suppresses human T lymphocyte pro-liferation through IFN-gamma-dependent and IFN-gamma-independent induction of apoptosis. J Immunol 1999 Oct 15;163(8):4182–91.

31. Dietlin TA, Hofman FM, Gilmore W, Stohlman SA, van der Veen RC. T cell expansion is regulated by activated Gr-1+ spleno-cytes. Cellular Immunol 2005 May;235(1):39–45.

32. Staykova MA, Berven LA, Cowden WB, Willenborg DO, Crouch MF. Nitric oxide induces polarization of actin in encepha-litogenic T cells and inhibits their in vitro trans-endothelial migration in a p70S6 kinase-independent manner. FASEB J 2003 Jul;17(10):1337–9.

33. Sun Y, Liu J, Qian F, Xu Q. Nitric oxide inhibits T cell adhesion and migration by down-regulation of beta1-integrin expression in immunologically liver-injured mice. Intern Immunopharmacol 2006 Apr;6(4):616–26.

Copyright of Immunopharmacology & Immunotoxicology is the property of Taylor & Francis Ltd and its

content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's

express written permission. However, users may print, download, or email articles for individual use.