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Anti-brain but not celiac disease antibodies in Landau-Kleffner Syndrome and related epilepsies
Journal of Neuroimmunology 160 (2005) 228 – 232 www.elsevier.com/locate/jneuroim
Short communication
Anti-brain but not celiac disease antibodies in Landau-Kleffner Syndrome and related epilepsies S. Boscoloa, V. Baldasb, G. Gobbic,1, L. Giordanod,1, G. Cionie,1, T. Notb, A. Venturab, E. Tongiorgia,* a
BRAIN Centre for Neuroscience, Department of Biology, University of Trieste, Via L. Giorgieri 10-34127, Trieste, Italy b Pediatric Clinic and Transfusional centre of the IRCCS Burlo Garofolo, Via dell’Istria 65, Trieste, Italy c U.O.NPI Dep. Materno Infantile Ospedale Maggiore, L.go Nigrisoli 2, Bologna, Italy d Neurological Pediatric Clinic, Spedali Riuniti, P.le Spedali Civili 1, Brescia, Italy e Dpt. Developmental Neuroscience, Stella Maris Scientific Institute, University of Pisa, Via dei Giacinti, 2, Italy Received 7 July 2004; received in revised form 22 October 2004; accepted 22 October 2004
Abstract The Landau-Kleffner syndrome, the continuous spikes-waves during slow sleep syndrome and the benign epilepsy of childhood with rolandic spikes are rare childhood epilepsies with unknown etiology. Improvement in patients treated with immunoglobulin suggests an involvement of the immune system. We provide immunohistochemical evidence of autoantibodies against rat brain auditory cortex, brainstem and cerebellum, in children suffering with one or more of these syndromes. Only 1/14 patient was celiac. D 2004 Elsevier B.V. All rights reserved. Keywords: Autoimmunity; Pediatric epilepsy; Anti-brain autoantibodies
1. Introduction The Landau-Kleffner syndrome (LKS), the continuous spikes-waves during slow sleep syndrome (CSWS) and the benign epilepsy of childhood with rolandic spikes (BRE) are distinct syndromes (ILAE, 1989). However, these epileptic disorders may be considered as part of a continuous pathological spectrum with BRE as a mild disorder, on one end, and CSWS as a more severe disorder, on the other end. The LKS, is a rare, age-related (onset 4–7 years) epileptic aphasia occurring in children who lose previously acquired speech and language abilities (Landau and Kleffner, 1957) and may occur without seizures being recorded. LKS Variant (LKSV) includes behavioral features of autistic
* Corresponding author. Tel.: +39 040 558 3864; fax: +39 040 568855. E-mail address: [email protected] (E. Tongiorgi). 1 These authors have equally contributed. 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.031
regression (Mantovani, 2000). All patients with LandauKleffner syndrome show, at some time during its course, the sleep-EEG pattern of continuous spike and wave discharges (Tassinari et al., 2002). CSWS is characterized by variable clinical manifestations and an EEG consisting of an almost continuous presence of spike waves discharges in slow sleep, persisting in three or more records over a period of at least one month. On the other hand, the EEG pattern consisting of CSWS may simply be a transient phase which influences the long outcome of the BRE and LKS (Veggiotti et al., 1999) and which characterizes the LKSV. BRE occurs in neurologically and cognitively normal children who usually present with a nocturnal partial seizure and with typical EEG showing centrotemporal and multifocal or generalized sharp slow waves (Beaussart, 1972). BRE accounts for 6–16% of all non-febrile seizures in children and, in 40% of patients, has a familial occurrence. All these epileptic syndromes appear in young children in the absence of obvious structural lesions suggesting that
S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
they might result from abnormalities occurring during brain maturation. The pathogenesis, optimal treatment and prognosis of these disorders are still unclear. An autoimmune etiology has been proposed in some LKS and LKSV patients because of the presence, in sera and cerebrospinal fluid, of autoantibodies to central and peripheral myelin, cell nucleus and blood vessels (Connolly et al., 1999). This
229
hypothesis is supported by the successful use of i.v. immunoglobulin as initial monotherapy of LKS (Mikati et al., 2002). The aim of this study was to detect the anti-brain reactivity of sera from patients suffering from one or more of these childhood conditions. In addition, since the labeling of the blood vessel previously described (Connolly et al.,
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
Fig. 1. Serum IgGs staining on sagittal rat brain sections. Immunohistochemical staining of serum IgGs (sera dilution 1:600) on rat Purkinje cells (A, D, G, J, M), rat auditory cortex layer V (B, E, H, K, N) and rat brainstem neurons (C, F, I, L, O). The healthy donor serum (A, B, C) is representative of the healthy donors that showed no staining. The CSWS patient sera (D, E, F), in analogy with the healthy donors, gave no staining. The LKSV without CSWS serum (G, H, I) showed a weak cytoplasmic staining (borderline) and a positive labeling of the nuclei (arrows) of neurons from the three brain areas considered. The LKSV with CSWS sera (J, K, L) produced a borderline staining on the cytoplasm of neurons from the three brain areas shown, while the partial epilepsy with CSWS serum (M, N, O) was clearly positive on the cytoplasm of neurons from cerebellum and brainstem and borderline on cortex neurons. Calibration bar=50 Am.
230
S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
donors. The mean patients age was 8.6 years (range 3–15 years), the healthy subjects mean age was 7 years (range 3– 7 years). Clinical diagnoses were assigned after examination of developmental and seizure history, neurological findings and EEG recordings. The patient group including LKS, CSWS and BRE, was diagnosed according to the international classification of epileptic syndromes (ILAE, 1989). The diagnosis of LKS was assigned to children who displayed a regression of previously normal language skills. The diagnosis of LKS-Variant was assigned following Echenne et al.’s (1992) suggestions. Sera were collected at the Hospitals of Trieste, Bologna, Pisa and Brescia (Italy) and were immediately stored at 80 8C. Informed consent was obtained from children’s parents.
1999) was reminiscent of the labeling produced by antitransglutaminase antibodies (Pratesi et al., 1998), we tested the hypothesis that these pediatric epilepsies might be one of the many neurological manifestations of celiac disease.
2. Methods 2.1. Patients This study included 14 epileptic patients (n=2 CSWS, n=3 LKS without CSWS; n=1 LKSV without CSWS, n=1 LKS with CSWS, n=2 LKSV with CSWS, n=3 BRE with CSWS, n=2 partial epilepsy with CSWS) and 16 healthy
A
Cerebellum 90% 80% 70% 60%
neg
50%
bord
40%
pos s pos
30% 20% 10% 0% Healthy donors n=16
B
CSWS n=2
LKS+LKSV n=4
CSWS+ n=8
Cortex V layer 90% 80% 70% 60%
neg
50%
bord
40%
pos s pos
30% 20% 10% 0% Healthy donors n=16
C
CSWS n=2
LKS+LKSV n=4
CSWS+ n=8
Brainstem 90% 80% 70% 60%
neg
50%
bord
40%
pos
30%
s pos
20% 10% 0% Healthy donors n=16
CSWS n=2
LKS+LKSV n=4
CSWS+ n=8
Fig. 2. Semi-quantitative densitometric analysis of sera IgG staining on rat cerebellum, cortex V layer and brainstem. The signal intensity on rat brain sections produced by the different sera was subdivided into four classes: negative (neg), borderline (bord), positive (pos), strongly positive (s pos), on the basis of the average pixel intensity recoded for the healthy subjects. On cerebellum (A) and cortex (B) most of healthy subjects resulted negative while the epileptic patients had border or positive densitometric values. The signal intensity increased on brainstem (C) both for healthy donors and epileptic patients, showing not only positive but also strongly positive values. However, in contrast to the healthy donors, none of the epileptic patients resulted negative on brainstem neurons.
S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
2.2. Celiac disease testing Anti-human tissue transglutaminase (h-tTG) immunoreactivity was tested in ELISA according to Sblattero et al. (2000). 2.3. Immunohistological testing Testing serum IgGs (1:600) on rat brain sections was performed in Trieste, Italy, according to previously published method (Hadjivassiliou et al., 2002). Semi-quantitative evaluation of the signal intensity on rat brain sections was performed with the image analysis software ImageProPlus (MediaCybernetics; Silver Spring, MD). The mean value F1 SD, of the darkest pixel intensity recoded on sections labeled with serum IgGs from 114 healthy donors was used as a reference value (negative). Borderline signal intensities included values between +1 and +2 SD; positive values were between +2 and +3 SD; strong positive values were above +3 SD. Fisher exact test has been carried out for each group/area with respect to the corresponding areas for the healthy subject group (significance pb0.05).
3. Results To determine the presence of anti-brain autoantibodies, the serum IgGs of the 16 healthy donors and the 14 epileptic children described above, were tested by immunohistochemistry on sagittal rat brain sections. Three representative brain regions, cerebellum (molecular layer, Purkinje cells and granule cell layer), auditory cortex (layer V) and brainstem are shown in Fig. 1. The staining levels on these selected brain areas were measured by semi-quantitative densitometry (Fig. 2, Table 1). Among the 16 healthy donors, 14 (87.5%) were negative or borderline for both cortex and cerebellum while 5 (31%) were positive or strongly positive for the brainstem; thus, 69% of healthy subjects were negative or borderline over the three brain areas. In analogy with the healthy donors, both the CSWS patient sera were negative or borderline on cerebellum and cortex and positive or strongly positive on brainstem. Of the four LKS-LKSV patients, one LKSV patient (25%) was positive on the nucleoli of Purkinje cells and was also positive or strongly positive on neurons from
231
brainstem and cortex (V layer), while the three LKS patients were positive on brainstem and one of them also on the cortex. When considered together, 25% LKS-LKSV patients were positive for the cerebellum, 100% were positive or strongly positive for the brainstem and 50% were positive for the cortical layer V. When CSWS were associated with LKS-LKSV or other types of epilepsy including BRE (defined as CSWS+ group), we always observed (8/8) a peculiar cytoplasmic staining in Purkinje cells that was absent in 14/16 healthy donors and in 6/6 other patients analyzed. However, according to the densitometric analysis, this cytoplasmic Purkinje cells staining was borderline in 5/8 CSWS+ patients and positive in 3/8 (37.5%; see also Fig. 1). The CSWS+ group resulted also positive or strongly positive on brainstem neurons (6/8; 75%) and cortex (2/8; 25%). In addition, 7/7 patients with LKS-LKSV with or without CSWS showed labeling of microvessels. In summary (Table 1), the cerebellum was stained by 6.5% of the healthy donors and by 28.5% of the epileptic patients, the brainstem by 31% of the healthy donors and by 85.5% of the epileptic syndrome patients while the cortex was labeled by 12.5% of the healthy donors and by 28.5% of the epileptic patients. The statistical analysis, despite the limited number of patients, showed significant differences on the brainstem. Finally, to test the hypothesis of whether these conditions are associated with celiac disease or not, an ELISA screening for anti-transglutaminase antibodies, the main autoantigen of celiac disease, was carried out. The ELISA test revealed just one positive patient (LKS with CSWS), for IgAs only.
4. Discussion This study shows that heightened anti-brain immunological response occurred significantly more frequently in patients affected by pediatric epilepsies including LKS, CSWS and BRE with respect to age-matched healthy subjects. However, these pathologies were not associated with celiac disease. Interestingly, patients with the CSWS syndrome did not have significant levels of anti-brain antibodies, but when EEG condition consisting on CSWS was associated with LKS, Rolandic Epilepsy or partial
Table 1 Relative frequency of positive/strong positive sera on the different rat brain areas Cerebellum Cortex V layer Brainstem a b c d
Healthy donors, n=16
CSWS, n=2
LKS/LKSV, n=4
CSWS+, n=8
Tot epilepsya, n=14
n=1 (6.3%) n=2 (12.5%) n=5 (31.3%)
n=0 (0%) n=0 (0%) n=2 (100%)
n=1 (25%) n=2 (50%) n=4 (100%)b
n=3 (37.5%) n=2 (25%) n=6 (75%)c
n=4 (28.6%) n=4 (28.6%) n=12 (85.7%)d
Total epilepsy is the cumulative percentage for the CSWS, LKS/LKSK and CSWS+ groups pooled together. Fisher test p=0.026. Fisher test p=0.055. Fisher test p=0.004.
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epilepsy, we always found a typical staining, with variable intensity, on the cytoplasm of Purkinje cells and other neurons. Furthermore, in agreement with previous studies (Connolly et al., 1999), the patient group including LKS and LKSV without CSWS, showed significant labeling of neuronal nuclei and small blood vessels. Interestingly, 28.5% of all epileptic patients (but 50% LKS/LKSV patients and 25% the CSWS+) label the soma and the apical dendrites of large pyramidal neurons from layer V of several cortical areas including the auditory cortex. It has been postulated that an impairment of the normal functioning of the connections from the auditory cortex to the thalamus might be the possible cause of acquired aphasia observed in LKS/LKSV children. In conclusion, our results support the hypothesis that, at least in some patients, an autoimmune mechanism might be involved in the aetio-pathogenesis of the LKS and CSWS related pediatric epilepsies.
Acknowledgements AV and ET were supported by the Telethon Foundation Grant E.1270 and Fondazione Cassa di Risparmio CRTrieste.
References Beaussart, M., 1972. Benign epilepsy of children with Rolandic (centrotemporal) paroxysmal foci. A clinical entity. Study of 221 cases. Epilepsia 13, 795 – 811.
Connolly, A.M., Chez, M.G., Pestronk, A., Arnold, S.T., Mehta, S., Deuel, R.K., 1999. Serum autoantibodies to brain in Landau-Kleffner variant, autism, and other neurologic disorders. J. Pediatr. 134, 607 – 613. Echenne, B., Cheminal, R., Rivier, F., Negre, C., Touchon, J., Billiard, M., 1992. Epileptic electroencephalographic abnormalities and developmental dysphasias: a study of 32 patients. Brain Dev. 14, 216 – 225. Hadjivassiliou, M., Boscolo, S., Davies-Jones, G.A., Grunewald, R.A., Not, T., Sanders, D.S., Simpson, J.E., Tongiorgi, E., Williamson, C.A., Woodroofe, N.M., 2002. The humoral response in the pathogenesis of gluten ataxia. Neurology 58, 1221 – 1226. ILAE, 1989. Proposal for revised classification of epilepsies and epileptic syndromes. Commission on Classification and Terminology of the International League Against Epilepsy. Epilepsia 30, 389 – 399. Landau, W.M., Kleffner, F.R., 1957. Syndrome of acquired aphasia with convulsive disorder in children. Neurology 7, 523 – 530. Mantovani, J.F., 2000. Autistic regression and Landau-Kleffner syndrome: progress or confusion? Dev. Med. Child Neurol. 42, 349 – 353. Mikati, M.A., Saab, R., Fayad, M.N., Choueiri, R.N., 2002. Efficacy of intravenous immunoglobulin in Landau-Kleffner syndrome. Pediatr. Neurol. 26, 298 – 300. Pratesi, R., Gandolfi, L., Friedman, H., Farage, L., de Castro, C.A., Catassi, C., 1998. Serum IgA antibodies from patients with coeliac disease react strongly with human brain blood-vessel structures. Scand. J. Gastroenterol. 33, 817 – 821. Sblattero, D., Berti, I., Trevisiol, C., Marzari, R., Tommasini, A., Bradbury, A., Fasano, A., Ventura, A., Not, T., 2000. Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease. Am. J. Gastroenterol. 95, 1253 – 1257. Tassinari, C.A., Rubboli, G., Volpi, L., Billard, C., Bureau, M., 2002. Electrical status epilepticus during slow sleep (ESES or CSWS) including acquired epileptic aphasia (Landau-Kleffner syndrome). In: Roger, J., Bureau, M., Dravet, C.H., Genton, P., Tassinari, C.A., Wolf, P. (Eds.), Epileptic Syndromes in Infancy, Childhood and Adolescence, 3rd ed. John Libbey, Eastleigh, pp. 265 – 283. Veggiotti, P., Beccaria, F., Guerrini, R., Capovilla, G., Lanzi, G., 1999. Continuous spike-and-wave activity during slow-wave sleep: syndrome or EEG pattern? Epilepsia 40, 1593 – 1601.
Short communication
Anti-brain but not celiac disease antibodies in Landau-Kleffner Syndrome and related epilepsies S. Boscoloa, V. Baldasb, G. Gobbic,1, L. Giordanod,1, G. Cionie,1, T. Notb, A. Venturab, E. Tongiorgia,* a
BRAIN Centre for Neuroscience, Department of Biology, University of Trieste, Via L. Giorgieri 10-34127, Trieste, Italy b Pediatric Clinic and Transfusional centre of the IRCCS Burlo Garofolo, Via dell’Istria 65, Trieste, Italy c U.O.NPI Dep. Materno Infantile Ospedale Maggiore, L.go Nigrisoli 2, Bologna, Italy d Neurological Pediatric Clinic, Spedali Riuniti, P.le Spedali Civili 1, Brescia, Italy e Dpt. Developmental Neuroscience, Stella Maris Scientific Institute, University of Pisa, Via dei Giacinti, 2, Italy Received 7 July 2004; received in revised form 22 October 2004; accepted 22 October 2004
Abstract The Landau-Kleffner syndrome, the continuous spikes-waves during slow sleep syndrome and the benign epilepsy of childhood with rolandic spikes are rare childhood epilepsies with unknown etiology. Improvement in patients treated with immunoglobulin suggests an involvement of the immune system. We provide immunohistochemical evidence of autoantibodies against rat brain auditory cortex, brainstem and cerebellum, in children suffering with one or more of these syndromes. Only 1/14 patient was celiac. D 2004 Elsevier B.V. All rights reserved. Keywords: Autoimmunity; Pediatric epilepsy; Anti-brain autoantibodies
1. Introduction The Landau-Kleffner syndrome (LKS), the continuous spikes-waves during slow sleep syndrome (CSWS) and the benign epilepsy of childhood with rolandic spikes (BRE) are distinct syndromes (ILAE, 1989). However, these epileptic disorders may be considered as part of a continuous pathological spectrum with BRE as a mild disorder, on one end, and CSWS as a more severe disorder, on the other end. The LKS, is a rare, age-related (onset 4–7 years) epileptic aphasia occurring in children who lose previously acquired speech and language abilities (Landau and Kleffner, 1957) and may occur without seizures being recorded. LKS Variant (LKSV) includes behavioral features of autistic
* Corresponding author. Tel.: +39 040 558 3864; fax: +39 040 568855. E-mail address: [email protected] (E. Tongiorgi). 1 These authors have equally contributed. 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.031
regression (Mantovani, 2000). All patients with LandauKleffner syndrome show, at some time during its course, the sleep-EEG pattern of continuous spike and wave discharges (Tassinari et al., 2002). CSWS is characterized by variable clinical manifestations and an EEG consisting of an almost continuous presence of spike waves discharges in slow sleep, persisting in three or more records over a period of at least one month. On the other hand, the EEG pattern consisting of CSWS may simply be a transient phase which influences the long outcome of the BRE and LKS (Veggiotti et al., 1999) and which characterizes the LKSV. BRE occurs in neurologically and cognitively normal children who usually present with a nocturnal partial seizure and with typical EEG showing centrotemporal and multifocal or generalized sharp slow waves (Beaussart, 1972). BRE accounts for 6–16% of all non-febrile seizures in children and, in 40% of patients, has a familial occurrence. All these epileptic syndromes appear in young children in the absence of obvious structural lesions suggesting that
S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
they might result from abnormalities occurring during brain maturation. The pathogenesis, optimal treatment and prognosis of these disorders are still unclear. An autoimmune etiology has been proposed in some LKS and LKSV patients because of the presence, in sera and cerebrospinal fluid, of autoantibodies to central and peripheral myelin, cell nucleus and blood vessels (Connolly et al., 1999). This
229
hypothesis is supported by the successful use of i.v. immunoglobulin as initial monotherapy of LKS (Mikati et al., 2002). The aim of this study was to detect the anti-brain reactivity of sera from patients suffering from one or more of these childhood conditions. In addition, since the labeling of the blood vessel previously described (Connolly et al.,
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
Fig. 1. Serum IgGs staining on sagittal rat brain sections. Immunohistochemical staining of serum IgGs (sera dilution 1:600) on rat Purkinje cells (A, D, G, J, M), rat auditory cortex layer V (B, E, H, K, N) and rat brainstem neurons (C, F, I, L, O). The healthy donor serum (A, B, C) is representative of the healthy donors that showed no staining. The CSWS patient sera (D, E, F), in analogy with the healthy donors, gave no staining. The LKSV without CSWS serum (G, H, I) showed a weak cytoplasmic staining (borderline) and a positive labeling of the nuclei (arrows) of neurons from the three brain areas considered. The LKSV with CSWS sera (J, K, L) produced a borderline staining on the cytoplasm of neurons from the three brain areas shown, while the partial epilepsy with CSWS serum (M, N, O) was clearly positive on the cytoplasm of neurons from cerebellum and brainstem and borderline on cortex neurons. Calibration bar=50 Am.
230
S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
donors. The mean patients age was 8.6 years (range 3–15 years), the healthy subjects mean age was 7 years (range 3– 7 years). Clinical diagnoses were assigned after examination of developmental and seizure history, neurological findings and EEG recordings. The patient group including LKS, CSWS and BRE, was diagnosed according to the international classification of epileptic syndromes (ILAE, 1989). The diagnosis of LKS was assigned to children who displayed a regression of previously normal language skills. The diagnosis of LKS-Variant was assigned following Echenne et al.’s (1992) suggestions. Sera were collected at the Hospitals of Trieste, Bologna, Pisa and Brescia (Italy) and were immediately stored at 80 8C. Informed consent was obtained from children’s parents.
1999) was reminiscent of the labeling produced by antitransglutaminase antibodies (Pratesi et al., 1998), we tested the hypothesis that these pediatric epilepsies might be one of the many neurological manifestations of celiac disease.
2. Methods 2.1. Patients This study included 14 epileptic patients (n=2 CSWS, n=3 LKS without CSWS; n=1 LKSV without CSWS, n=1 LKS with CSWS, n=2 LKSV with CSWS, n=3 BRE with CSWS, n=2 partial epilepsy with CSWS) and 16 healthy
A
Cerebellum 90% 80% 70% 60%
neg
50%
bord
40%
pos s pos
30% 20% 10% 0% Healthy donors n=16
B
CSWS n=2
LKS+LKSV n=4
CSWS+ n=8
Cortex V layer 90% 80% 70% 60%
neg
50%
bord
40%
pos s pos
30% 20% 10% 0% Healthy donors n=16
C
CSWS n=2
LKS+LKSV n=4
CSWS+ n=8
Brainstem 90% 80% 70% 60%
neg
50%
bord
40%
pos
30%
s pos
20% 10% 0% Healthy donors n=16
CSWS n=2
LKS+LKSV n=4
CSWS+ n=8
Fig. 2. Semi-quantitative densitometric analysis of sera IgG staining on rat cerebellum, cortex V layer and brainstem. The signal intensity on rat brain sections produced by the different sera was subdivided into four classes: negative (neg), borderline (bord), positive (pos), strongly positive (s pos), on the basis of the average pixel intensity recoded for the healthy subjects. On cerebellum (A) and cortex (B) most of healthy subjects resulted negative while the epileptic patients had border or positive densitometric values. The signal intensity increased on brainstem (C) both for healthy donors and epileptic patients, showing not only positive but also strongly positive values. However, in contrast to the healthy donors, none of the epileptic patients resulted negative on brainstem neurons.
S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
2.2. Celiac disease testing Anti-human tissue transglutaminase (h-tTG) immunoreactivity was tested in ELISA according to Sblattero et al. (2000). 2.3. Immunohistological testing Testing serum IgGs (1:600) on rat brain sections was performed in Trieste, Italy, according to previously published method (Hadjivassiliou et al., 2002). Semi-quantitative evaluation of the signal intensity on rat brain sections was performed with the image analysis software ImageProPlus (MediaCybernetics; Silver Spring, MD). The mean value F1 SD, of the darkest pixel intensity recoded on sections labeled with serum IgGs from 114 healthy donors was used as a reference value (negative). Borderline signal intensities included values between +1 and +2 SD; positive values were between +2 and +3 SD; strong positive values were above +3 SD. Fisher exact test has been carried out for each group/area with respect to the corresponding areas for the healthy subject group (significance pb0.05).
3. Results To determine the presence of anti-brain autoantibodies, the serum IgGs of the 16 healthy donors and the 14 epileptic children described above, were tested by immunohistochemistry on sagittal rat brain sections. Three representative brain regions, cerebellum (molecular layer, Purkinje cells and granule cell layer), auditory cortex (layer V) and brainstem are shown in Fig. 1. The staining levels on these selected brain areas were measured by semi-quantitative densitometry (Fig. 2, Table 1). Among the 16 healthy donors, 14 (87.5%) were negative or borderline for both cortex and cerebellum while 5 (31%) were positive or strongly positive for the brainstem; thus, 69% of healthy subjects were negative or borderline over the three brain areas. In analogy with the healthy donors, both the CSWS patient sera were negative or borderline on cerebellum and cortex and positive or strongly positive on brainstem. Of the four LKS-LKSV patients, one LKSV patient (25%) was positive on the nucleoli of Purkinje cells and was also positive or strongly positive on neurons from
231
brainstem and cortex (V layer), while the three LKS patients were positive on brainstem and one of them also on the cortex. When considered together, 25% LKS-LKSV patients were positive for the cerebellum, 100% were positive or strongly positive for the brainstem and 50% were positive for the cortical layer V. When CSWS were associated with LKS-LKSV or other types of epilepsy including BRE (defined as CSWS+ group), we always observed (8/8) a peculiar cytoplasmic staining in Purkinje cells that was absent in 14/16 healthy donors and in 6/6 other patients analyzed. However, according to the densitometric analysis, this cytoplasmic Purkinje cells staining was borderline in 5/8 CSWS+ patients and positive in 3/8 (37.5%; see also Fig. 1). The CSWS+ group resulted also positive or strongly positive on brainstem neurons (6/8; 75%) and cortex (2/8; 25%). In addition, 7/7 patients with LKS-LKSV with or without CSWS showed labeling of microvessels. In summary (Table 1), the cerebellum was stained by 6.5% of the healthy donors and by 28.5% of the epileptic patients, the brainstem by 31% of the healthy donors and by 85.5% of the epileptic syndrome patients while the cortex was labeled by 12.5% of the healthy donors and by 28.5% of the epileptic patients. The statistical analysis, despite the limited number of patients, showed significant differences on the brainstem. Finally, to test the hypothesis of whether these conditions are associated with celiac disease or not, an ELISA screening for anti-transglutaminase antibodies, the main autoantigen of celiac disease, was carried out. The ELISA test revealed just one positive patient (LKS with CSWS), for IgAs only.
4. Discussion This study shows that heightened anti-brain immunological response occurred significantly more frequently in patients affected by pediatric epilepsies including LKS, CSWS and BRE with respect to age-matched healthy subjects. However, these pathologies were not associated with celiac disease. Interestingly, patients with the CSWS syndrome did not have significant levels of anti-brain antibodies, but when EEG condition consisting on CSWS was associated with LKS, Rolandic Epilepsy or partial
Table 1 Relative frequency of positive/strong positive sera on the different rat brain areas Cerebellum Cortex V layer Brainstem a b c d
Healthy donors, n=16
CSWS, n=2
LKS/LKSV, n=4
CSWS+, n=8
Tot epilepsya, n=14
n=1 (6.3%) n=2 (12.5%) n=5 (31.3%)
n=0 (0%) n=0 (0%) n=2 (100%)
n=1 (25%) n=2 (50%) n=4 (100%)b
n=3 (37.5%) n=2 (25%) n=6 (75%)c
n=4 (28.6%) n=4 (28.6%) n=12 (85.7%)d
Total epilepsy is the cumulative percentage for the CSWS, LKS/LKSK and CSWS+ groups pooled together. Fisher test p=0.026. Fisher test p=0.055. Fisher test p=0.004.
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S. Boscolo et al. / Journal of Neuroimmunology 160 (2005) 228–232
epilepsy, we always found a typical staining, with variable intensity, on the cytoplasm of Purkinje cells and other neurons. Furthermore, in agreement with previous studies (Connolly et al., 1999), the patient group including LKS and LKSV without CSWS, showed significant labeling of neuronal nuclei and small blood vessels. Interestingly, 28.5% of all epileptic patients (but 50% LKS/LKSV patients and 25% the CSWS+) label the soma and the apical dendrites of large pyramidal neurons from layer V of several cortical areas including the auditory cortex. It has been postulated that an impairment of the normal functioning of the connections from the auditory cortex to the thalamus might be the possible cause of acquired aphasia observed in LKS/LKSV children. In conclusion, our results support the hypothesis that, at least in some patients, an autoimmune mechanism might be involved in the aetio-pathogenesis of the LKS and CSWS related pediatric epilepsies.
Acknowledgements AV and ET were supported by the Telethon Foundation Grant E.1270 and Fondazione Cassa di Risparmio CRTrieste.
References Beaussart, M., 1972. Benign epilepsy of children with Rolandic (centrotemporal) paroxysmal foci. A clinical entity. Study of 221 cases. Epilepsia 13, 795 – 811.
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