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1 Acta Neurol Scand 2004 DOI: /j x Printed in UK. All rights reserved IgA antibodies against gliadin and gluten in multiple sclerosis Multiple sclerosis (MS) is a debilitating neurological disease without known cause. However, immunological changes found in this disease are thought to reflect an autoimmune reaction (1, 2) in genetically susceptible individuals (3). Antibodies that bind to brain basic myelin protein have been detected, and peptides from myelin basic protein can induce experimental demyelinating encephalitis in animal models. As immunoglobulin A (IgA) antibodies in serum against gliadin and gluten could be demonstrated in gluten-induced cerebellar ataxia with peripheral neuritis and ataxia (4), and as these antibodies have a high affinity for the brain barrier vasculature (5), we decided to measure these antibodies in serum also in MS. Patients and methods A N E B The patients were obtained through the Multiple Sclerosis Society of Norway, which is thanked for their outstanding efforts. All the patients volunteered after having received an informed letter describing why and how the examination was going to be performed. The patients all had their diagnosis determined by the neurological departments of the University Dispatch: Journal: ANE CE: Hari Journal Name Manuscript No. Author Received: No. of pages: 3 PE: Shyamala Reichelt K-L, Jensen D. IgA antibodies against gliadin and gluten in multiple sclerosis. Acta Neurol Scand 2004 DOI: /j x Ó Blackwell Munksgaard Background Multiple changes in antibodies against various antigens are found in multiple sclerosis (MS). Objective We wanted to measure immunoglobulin A (IgA) antibodies to some common food antigens in MS and also IgG against gliadin and gluten. Methods The IgA antibodies were measured in serum against gluten, gliadin, lactoglobulin, lactalbumin, casein and ovalbumin in patients with MS and controls using ELISA technique. IgG was likewise measured for gluten and gliadin. Results Highly significant increases compared with controls were found for IgA and IgG antibodies against gliadin and gluten. IgA antibodies against casein were significantly increased. Anti-endomycium and anti-transglutaminase antibodies were negative. Conclusions The data presented indicates that there may be a possible moderately increased uptake of some specific proteins from the gut in MS compared with controls. Copyright Ó Blackwell Munksgaard 2004 ACTA NEUROLOGICA SCANDINAVICA K.-L. Reichelt 1, D. Jensen 2 1 Institute of Pediatric Research; 2 Department of Neurology, University of Oslo, Oslo, Norway Key words : immunoglobulin A; antibodies; multiple sclerosis; gliadin; gluten; casein K-L Reichelt, Institute of Pediatric Research, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway Tel.: Accepted for publication 14 April, 2004 of Oslo at either Ullevål City Hospital or The National Hospital (Rikshospitalet) [Poser criteria]. None were first episode patients and had experienced from two or more worsening episodes with at least two separate lesions (intermittent relapsing type). Those included in the study had typical oligoclonal immunoglobulin band in the CSF and half had been examined by NMR. Two had an unremitting course but of slow worsening (progressive subtype). The total number of patients were 36 with an age range of years (median 44 years). Of them, 21 were females and 15 males. We do not have the HLA classification on these patients collected systematically. Normal controls (n ¼ 26) were obtained through Fu rsts laboratory in Oslo (Clinical Chemistry Laboratory of high standard) were of the age range years (median 38 years). Of them, 16 were females and 8 males. Excluding the five oldest persons in the patient group older than the oldest control did not have any impact on the results. Venous blood was obtained by venous tap and the IgA antibodies measured by ELISA technique as described (6, 7). Costar ERIA microplates 2(no. 3590) were coated with antigens at concentrations found optimal for different antigens ( g/l), and the patientsõ sera were tested at a ratio 1

2 Reichelt & Jensen of 1:400. The measurements were taken at Fu rsts laboratory on a commercial basis but blinded. These tests are part of the celiac screening battery run routinely. Known standards were run with each analytical series. Whenever values were above the cut-off for celiac disease, endomycium and lately anti-trans-glutaminase antibodies were run. Statistics As the distribution of the IgA antibodies is extremely skewed (not parametrically distributed) we chose to use nonparametric statistics: the Mann Whitney U-test, two-tailed. Results The results of the measurements are illustrated in Table 1. We thus obtained the following statistics: gluten against control showed serum IgA antibodies that were higher than controls with P < (two-tailed) and statistical characteristic with U ¼ and U ¼ IgA antibodies against gliadin (Table 2) also had P < (U ¼ 88 and U ¼ 824). For casein P-value (two-tailed) was (U ¼ 322 and U ¼ 614). immunoglobulin A antibodies against lactoglobulin, lactalbumin and ovalbumin did not show statistical increases. However, one patient had IgA Table 1 Serum IgA antibody levels in multiple sclerosis (MS) and controls Mean Median Number 95% CI P-value MS IgA gliadin <0.001 IgA gluten <0.001 IgA casein Control IgA gliadin IgA gluten IgA casein P-values using Mann Whitney U-test for patient data against controls. Six of 38 patients had IgA values against gliadin and gluten above the cutoff for celiac disease, while none of the controls showed such increases. Counting numbers with IgA antibody levels above the cutoff for celiac disease, we find chi-square values with P < Table 2. IgG antibodies against gluten and gliadin in MS Mean Median Number 95% CI P-value MS Gliadin <0.001 Gluten <0.001 Control Gliadin Gluten antibodies against ovalbumin six times higher than the upper cut-off value. Total IgA levels were in the normal range (not shown), although two of the 28 patients had no demonstrable IgA antibodies against the specific proteins tested. The trans-glutaminase test was negative also for those with IgA levels in serum above the cutoff for celiac disease, however, celiac disease is unlikely. The IgG antibody levels against gluten were different from controls with P < (U ¼ 138 and U ¼ 773.5). For gliadin a P < was obtained (U ¼ 88 and U ¼ 824). There was no increase in IgA antibodies against beta-lactoglobulin, lact-albumin and ovalbumin (not shown). Discussion As MS is found mainly in the temperate zone, which is also the dairy farming sector of the world, we expected that the possible specific IgA increase to be against casein. However, the highly significant differences from the controls were found in serum IgA antibodies against gliadin and gluten which was extremely significant (P < 0.001; Table 2). Increase in specific serum IgA antibodies is thought to reflect increased uptake of protein from the gut. In addition, normal persons take up proteins from the gut (8 10) as well as fragments of proteins such as peptides (11). Very many epitopes that induce antibody formation are indeed peptides. The fact that dietary proteins are found in mothers milk (12 14) further reflect this uptake. For MS patients in remission, no increase in antibodies against milk proteins were found (15), while in our case most of the patients if not all were in an active phase of their disease based on their subjective evaluation of worsening. Using a sheep erythrocytes agglutination assay, another group did not find antibody increase towards gluten and gliadin (16). This may be the result of methodical differences, but again might be caused by differences in disease process (not stated). Furthermore, a general antibody test instead of measuring specific subclasses, may, because of increase in one and decrease in another species of antibodies, mask specific changes. Gliadin is especially noxious as shown for cerebellar atrophy and ataxia (4), celiac disease and gluten/gliadin related epilepsy (17, 18).The longer one is exposed to gluten in celiac disease the greater is the risk of developing an autoimmune disorder (19). In celiac disease, IgA antibodies against gliadin and gluten are usually increased and these antibodies do have a high affinity for the blood brain barrier vasculature (5), probably altering the permeability of this barrier. ELISA measured IgG antibodies are as described (6, 7). Although not very high, the level was very significant compared with controls run simultaneously. 2

3 1 IgA antibodies against gliadin and gluten in MS Quite intriguing and possibly of relevance to MS, is the observation that chronic inflammatory bowel disease causes lesions in the white-matter of the brain (20, 21). Lesions of the gut would increase the permeability of the gut, and also the uptake of gluten and gliadin and thus increase the IgA antibodies to these proteins. Increased intestinal permeability has been reported in MS(22) as well as possible inflammatory bowel disease (23). On a purely speculative note, is it possible that the reported connection between MS and measles antibodies (24, 25) could be the result of a chronic gut inflammation by measles as in regressive autism (26), where genetic material from measles viruses has been found in active lymph nodes of the gut (27, 28). The IgA antibody increase may be secondary to an increased gut uptake or an epi-phenomenon. It could, however, also be a primary cause. Possibly relevant to this is that butyrophilin from milk, can induce anti-myelin oligo-dendrocyte glycoprotein antibodies and a CD4+ T-cell response in animals with Experimental autoimmune encephalitis (29). Conclusion Increased levels of IgA and IgG antibodies in serum against gluten and gliadin and possibly also for casein in MS patients may indicate a possible increased gut permeability to certain proteins. References 1. Bansil S, Cook SD, Rohowsky-Kochan C. Multiple sclerosis: immune mechanism and update on current therapies. Annal Neurol 1995;37: Hintzen RQ, Polman CH, Lucas CJ, vanlier RAW. Multiple sclerosis: immunological findings and possible implications for therapy. J Neuroimmunol 1992;39: Reindl M, Linington C, Brehm U et al. Antibodies against the myelin oligodendrocyte glycoprotein and the myelin basic protein in multiple sclerosis and other neurological diseases: a comparative study. Brain 1999;122: Hadjivassiliou M, Boscolo S, Davies-Jones GAB, et al. The humoral response in the pathogenesis of Gluten ataxia. Neurology 2002;58: Pratesi R, Gandolfi L, Friedman H, et al. Serum IgA antibodies from patients with celiac disease react strongly with human brain blood-vessel structures. Scand J Gastroenterol 1998;33: Scott H, Rognum TO, Midtvedt TJ, Brandtzaeg P. Age related changes in human serum antibodies to dietary and colonic bacteria antigens measured by enzyme-linked immunoabsorbent assay. Acta Pathol Microbiol Immunol Scand 1985a;93: Scott H, Rognum TO, Brandtzaeg P. Performance testing of antigen coated polystyrene micro-plates for ELISA measurements of serum antibodies to bacterial and dietary antigens. Acta Pathol Microbiol Immunol Scand 1985b;93: Paganelli R, Levinsky RJ. Solid phase radioimmunoassay for the detection of circulating food protein antigen in human serum. J Immunol Method 1980;37: Husby S, Jensenius JC, Svehag SE. Passage of undegraded dietary antigen into the blood of healthy adults. Scand J Immunol 1985;22: Gardner MLG. Absorption of intact proteins and peptides. In: Johnson LR, ed. Physiology of the gastrointestinal tract, 3rd edn. New York: Raven Press, 1994; Chabance B, Marteau P, Rambaud JC et al. Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie 1998;80: Kilshaw PJ, Cant AJ. The passage of maternal dietary protein into human breast milk. Int Arch Allergy Appl Immunol 1984; 75: Axelsson I, Jacobsson I, Lindberg T, Benediktsson U. Bovine beta-lactoglobulin in human milk. Acta Paediat Scand 1986;75: Troncone R, Scarcella A, Donatiello A et al. Passage of gliadin into human breast milk. Acta Paed Scand 1987;76: Monetini L, Cavallo MG, Manfrini S et al. Antibodies to bovine beta-casein in diabetes and other autoimmune diseases. Horm Metab Res 2002;34: Hunter AL, Rees BWG and Jones LT. Gluten antibodies in patients with multiple sclerosis. Hum Nutr Appl Nutr 1984;38A: Chapman RWG, Laidlow JM, Colin-Jones D et al. Increased prevalence of epilepsy in celiac disease. BMJ 1978;22: Gobbi G, Bouquet F, Gicco L et al. Coeliac disease, epilepsy, and cerebral calcifications. Lancet 1992;340: Ventura A, Ragazzu G, Greco L. Duration of exposure to gluten and risk for autoimmune disorders in patients with celiac disease. Gastroenterology 1999;117: Geissler A, Andus T, Roth M et al. Focal white-matter lesions in brain of patients with inflammatory bowel disease. Lancet 1995;345: Hart PE, Gould SR, MacSweeney et al. Brain white-matter lesions in inflammatory bowel disease. Lancet 1998;351: Yacyshyn B, Meddings J, Sadowksi D, Bowen-Yacyshyn MB. Multiple sclerosis patients have peripheral CD45RO+B Cells and increased intestinal permeability. Dig Dis Sci 1996;41: Vandvik B, Degre M. Measles virus antibodies in serum and cerebro-spinal fluid in patients with multiple sclerosis and other neurological disorders, with special reference to measles antibody synthesis within the central nervous system. J Neurol Sci 1975;24: Felgenhauer K, Schadlich HJ, Nekic M, Ackermann R. Cerebrospinal fluid virus antibodies. A diagnostic indicator for multiple sclerosis? J Neurol Sci 1985;71: Wakefield AJ, Anthony A, Murch SH et al. Enterocolitis in children with developmental disorders. Am J Gastroenterol 2000;95: Uhlmann V, Martin CM, Sheils O et al. Potential viral pathogenic mechanism for a new variant of inflammatory bowel disease. J Clin Mol Pathol 2002;55: Martin CM, Uhlmann V, Killalea A et al. Detection of measles virus in children with ileo-colonic lymphoid nodular hyperplasia, enterocolitis and developmental disorder. Mol Psychiat 2002;7:S Steerl A, Schubart A, Storch M et al. Butyrophilin, a milk protein, modulates the encephalitogenic T cell response to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis. J Immunol 2000;165:

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