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Address correspondence and reprint requests to Dr. R. Cimaz at Department of Pediatrics, Via Commenda 9, 20122 Milano, Italy. E-mail: Rolando.Cimaz@unimi.it
Summary: Purpose: To measure anti-cardiolipin (aCL), anti-β2 glycoprotein I (anti-β2GPI), and anti-prothrombin (aPT) antibodies in young patients with epilepsy, and to correlate their presence with demographic data, clinical diagnoses, laboratory and neuroradiologic findings, and antiepileptic drugs (AEDs).
Methods: Sera from one hundred forty-two consecutive patients with epilepsy with a median age of 10 years were tested for aCL and anti-β2GPI autoantibodies by solid-phase assays. aPT antibodies also were assayed in sera from 90 patients. Positive results were confirmed after a minimum of 6 weeks. Antinuclear antibodies (ANAs) and antibodies against extractable nuclear antigens (ENAs) also were tested.
Results: An overall positivity of 41 (28.8%) of 142 sera was found. Fifteen patients were positive for aCL, 25 for anti-β2GPI, and 18 for aPT antibodies. Several patients (12%) displayed more than one specificity in their serum. Only one of these patients had a concurrent positivity for ANAs and ENAs. A predominance of younger patients was found in the antibody-positive group. All types of epilepsy were represented in the positive group. No relation between antibody positivity and AEDs was found. Diffuse ischemic lesions at computed tomography (CT)/magnetic resonance imaging (MRI) scans were present in higher percentages in patients who were antibody positive. No positive patient had a history of previous thrombosis or other features related to systemic lupus erythematosus (SLE), and no patient was born of a mother with SLE.
Conclusions: Our study suggests a relation between epilepsy and aPL in young patients. A pathogenetic role for these autoantibodies cannot be excluded, and their determination might prove useful even from a therapeutic point of view.
Antiphospholipid (aPL) antibodies are a family of autoantibodies frequently present in the sera of patients with systemic lupus erythematosus (SLE) or other autoimmune diseases. The association between the presence of aPL and thrombosis (venous or arterial) or recurrent fetal losses is well known, and the clinical picture is referred to as the antiphospholipid syndrome (APS), isolated (primary APS or PAPS) or secondary to a connective tissue disease (1,2).
The spectrum of neurologic disorders associated with the presence of aPL in patients with primary or secondary APS is wide (3–7), and central nervous system involvement in APS is quite frequent in both the primary and secondary forms (8–11). Ischemic events have been described in aPL-positive adults and children (12–17). Moreover, an association between epilepsy and the presence of aPL has been described in studies performed in patients with SLE (18,19), unselected adult epilepsy patients (20,21), or children with partial seizures (22,23).
The antigen target of aPL is still a matter of debate. Rather than being directed against anionic phospholipids (PLs) alone, aPL appear to recognize anionic PL-binding proteins complexed with PL (24,25). Among them, β2-glycoprotein I (β2GPI) appears to play a major pathogenic role. Actually, endothelium-bound β2GPI offers suitable epitopes for circulating aPL (including anti-β2GPI antibodies), and antibody binding has been shown to induce an endothelial proinflammatory and procoagulant phenotype (26,27). Interestingly, our group recently demonstrated that human brain microvascular endothelial cells bind higher amounts of β2GPI and can be activated by immunoglobulin G (IgG) from aPL-positive sera and by anti-β2GPI antibodies (27). In addition, β2GPI was identified immunohistologically on brain cells (28), and functional central nervous system (CNS) involvement was reported in experimental models of APS (29).
There is now general agreement that prothrombin, another PL-binding protein, is also one of the target antigens of aPL (30). Specifically, it has been demonstrated that anti-prothrombin (aPT) antibodies display lupus anticoagulant (LA) activity, and that with anti-β2GPI antibodies, they are responsible for anticoagulant activity in the majority of LA-positive plasmas (31,32).
Altogether, these findings suggest a pathogenetic role for aPL even in epilepsy, by means of microinfarcts secondary to ischemic events, or by an immune-mediated mechanism directed against endothelial or neuronal cells, as suggested in other models (33,34). To clarify these issues, we studied prospectively the presence of aPL (in particular, anti-cardiolipin (aCL), anti-β2GPI, and aPT antibodies) in an unselected series of young patients with epilepsy, and tried to correlate antibody positivity with the clinical diagnosis, antinuclear antibody (ANA) status, neuroradiologic abnormalities, and drug treatment.
MATERIALS AND METHODS
The study was performed on unselected consecutive patients followed at the Seizure Clinics of the Centro Regionale per l'Epilessia Infantile, Ospedale Fatebenefratelli, Milano, and of Centro di Neuropsichiatria Infantile Mondino, Pavia, during 1997. Patients were eligible only if they had had the onset of epilepsy before the age of 18 years. Patients older than 25 years at their clinic visit were excluded. For all eligible patients, the informed consent of a parent or guardian was given for the use of their blood in this study. The research received approval by the institutions' ethical committees.
During routine venipuncture, 2–3 additional milliliters of blood was drawn, and immediately centrifuged. Sera were then frozen and kept at –20°C, until the assays were centrally performed. Epilepsy was classified according to the International Classification of Epilepsies and Epileptic syndromes (35). For each patient, a customized data sheet included demographic data and clinical characteristics such as diagnosis, age at onset of epilepsy, frequency of seizures, EEG abnormalities, medical treatment, neuroradiologic abnormalities, and complete blood count values. Relevant variables and laboratory results were then entered into a computerized spreadsheet. Descriptive statistics and comparison of results (t test or χ2, as indicated) were performed with Excel 6.0 for Windows 95.
Anti-cardiolipin antibodies (aCLs) were detected by a solid-phase enzyme-linked immunosorbent assay (ELISA) as described (36). In brief, plates were coated with CL (Sigma-Aldrich S.r.L., Milan, Italy; 50 μg/ml in ethanol) by evaporating overnight at 4°C. Plates were then blocked by 10% fetal calf serum (FCS; Sigma-Aldrich) –0.15 M phosphate-buffered saline (PBS), pH 7.4, for 2 h, washed 3 times with FCS-PBS, and then incubated with samples for 2 h. After further washes with FCS-PBS, p-nitrophenyl phosphate substrate was used to reveal IgG or IgM binding. Optical density (OD) values of the enzymatic reactions were read at 405 nm with a microplate photometer (Biorad Platereader Model; Bio-Rad Laboratoires, S.r.L., Milan, Italy). Values were expressed as GPL or MPL units, and sera were considered positive when >10 units.
Anti-β2GPI antibodies were detected by a solid-phase assay, as described (36). In brief, polystyrene irradiated microtiter plates (Combiplate Enhanced Binding; Labsystems, Helsinki, Finland) were coated with human β2GPI (10 μg/ml) in 0.05 M carbonate buffer, pH 9.6, overnight at 4°C. The plates were then blocked with 0.15 M PBS, pH 7.2, containing 1% BSA. The serum samples, filtered on a 0.2-μm filter (Nalgene Company, New York, U.S.A.) and diluted 1:50 in 0.05% Tween 20/PBS (PBS-Tween), were added (100 μl/well) and incubated for 2 h at room temperature. The plates were washed 3 times with PBS-Tween and incubated 2 h with a conjugated of alkaline phosphatase goat anti-human IgG or anti-human IgM antibodies, diluted in PBS/Tween. After three additional washings, p-nitrophenyl phosphate (Sigma Chemical Company) in 0.05 M Mg-carbonate buffer, pH 9.8, was added to develop the color. The OD was read at 405 nm in a Novapath Microplate Reader (BioRad Laboratories, Hercules, CA, U.S.A.). The OD of blank wells (no sample) was automatically subtracted. In each run, the same IgG-positive and IgM-positive sera were added at three different dilutions in PBS-Tween, spanning the entire range of positivity. The reaction was stopped when these three samples reached a predetermined reading. To check the specificity of the binding, anti-β2GPI positive sera (10 IgG and 10 IgM) as well as 10 normal human sera were tested on both coated and uncoated wells. The control group comprised 61 apparently healthy children at their regular routine preventive visits in the community-based health centers. The cut-off points were calculated as the mean value plus 2 standard deviations for the log OD values, as previously described (37).
Anti-prothrombin (aPT) antibodies were detected by calcium-containing a PT ELISA (38). In brief, polystyrene irradiated microtiter plates (Combiplate Enhanced Binding, Labsystems, Helsinki, Finland) were coated overnight at 4°C with 50 μl/well of 50 μg/ml human prothrombin (Diagnostica Stago, Asnières, France) in Tris-buffered saline (0.05 M Tris, 0.15 M NaCl, pH 7.4) containing 0.005 M CaCl2 (TBS-Ca). After washing 3 times with 200 μl TBS-Ca containing 0.1% Tween 20, wells were blocked for 1 h at room temperature with 150 μl/well of 1% BSA (A7906, Sigma-Aldrich) in TBS-Ca/0.1% Tween 20. After washing, wells were then incubated (1 h, 37°C) with 50 μl/well of samples diluted 1:50 in TBS-Ca containing 0.1% Tween 20 and 1% BSA. After new washing, plates were incubated (1 h, 37°C) with 50 μl/well of AP-conjugated goat anti-human IgG or IgM antibodies (A-3188, A-9794, Sigma-Aldrich) diluted 1:1,000 in blocking buffer. Finally, for color developing, 100 μl/well of 0.6 mg/ml p-nitrophenyl phosphate (Sigma-Aldrich) in diethanolamine buffer (pH 9.8) was added. OD measurements and determination of the cut-off values were performed as described for the anti-β2GPI ELISA. A standard curve was prepared from reference serum with a known high aPT activity.
Patients who were positive for aPL were recalled ≥ 6 weeks after the first sample was drawn, to repeat the test and confirm the positivity.
ANAs were detected by standard indirect immunofluorescence on Hep-2 cells, as previously described (39). ANA-positive sera (at starting dilution of 1:160 dilution) (40) were further assayed for antibodies directed against extractable nuclear antigens (ENAs) by commercial ELISA (Diamedix, Miami, FL, U.S.A.) and against double-stranded DNA by C. luciliae assay, as described (41).
A total of 142 patients were enrolled in this prospective study. Eighty-seven were male and 55 female patients. The median age at the moment of blood drawing was 10 years (range, 1–25 years), whereas the median age at the onset of epilepsy was 20 months (range, 0–15 years).
The cumulative results of IgG and IgM aPL assays in all patients studied are shown in Fig. 1. Fifteen (10.6%) of the 142 patients were positive for aCL. IgG aCL were found in 11 (7.7%), IgM aCL in two (1.4%), and both isotypes in two (1.4%) patients. According to the KAPS standardized international units (GPL/MPL units) (42), seven had medium and eight had low titers of either IgG or IgM aCL. A total of 25 (17.6%) of 142 patients were positive for anti-β2GPI. Seventeen (12.0%) patients had IgG anti-β2GPI, four (2.8%) IgM anti-β2GPI, and four (2.8%) patients had both isotypes of anti-β2GPI. Additionally, aPT antibodies were determined in 90 patients. The frequency of aPT positivity was 18 (20.0%) of 90); four (4.4%) patients had IgG aPT, 11 (12.2%) had IgM aPT, and three (3.3%) patients had both IgG and IgM isotypes of aPT. The overall aPL positivity (aCL, anti-β2GPI, and aPT) in all patients studied is shown in Fig. 2; as can be seen, almost 30% of patients with epilepsy were positive for at least one subtype of aPL. In all positive patients, the tests were repeated ≥6 weeks after the first sample was drawn, and in all cases, the positivities were confirmed.
Ten patients had a positive ANA (seven with a speckled and three with a homogeneous pattern), but only three of them had a concurrent positivity for aPL. Only one serum, from a girl with cryptogenetic generalized epilepsy with an onset at age 1 year, was simultaneously positive for aCL, ANA, and ENA (anti-SSA/Ro), whereas anti-double stranded DNA were absent.
As can be seen in Table 1, the comparison of the demographic data between antibody-positive and -negative patients showed no sex differences between the two groups but a predominance of younger patients in the positive group, with 43.9% patients in the first 5 years of life and >70% younger than 10 years old.
Table 1. Sex and age distribution among antiphospholipid antibody-positive and -negative patients
Positive n (%)
Negative n (%)
Total n (%)
The different diagnoses of patients positive or negative for aPL are shown in Table 2. Among the 41 positive patients, 19 (46.3%) had a diagnosis of symptomatic epilepsy, whereas others belonged to the group of idiopathic (9.8%) or cryptogenic (31.7%) epilepsy. In the group of antibody-negative patients, the three categories on the contrary were more equally represented, with 27.7% of cases being idiopathic, 30.7% cryptogenic, and 36.6% symptomatic.
Table 2. Epilepsy distribution between antiphospholipid antibody-positive and -negative patients
A neuroradiologic study [computed tomography (CT) scan and/or magnetic resonance imaging (MRI)] was available for the majority (117 of 142) of the patients in our study. Of the 41 antibody-positive patients, 35 had a recent scan available. The results are shown in Table 3. Of note, four of 35 positive patients and four of 82 negative patients who had a scan available showed diffuse ischemic lesions. These four antibody-positive patients had a diagnosis of symptomatic epilepsy, localized in one case and generalized in three cases. Perinatal anoxic damage was thought to be the cause of the disease in three cases, whereas one patient had postoperative complications after major heart surgery.
Table 3. Neuroradiological findings in 117 patients where recent CT or MRI was available
aPL- Positive patients n (%)
aPL- Negative patients n (%)
Total n (%)
CT, computed tomography; MRI, magnetic resonance imaging.
Diffuse ischemic lesions
Focal ischemic lesions
Diffuse inflammatory anomalies
Posthemorrhagic hydrocephalus sequelae
A recent EEG was available for all patients of this study. The EEG was normal in four (9.8%) of 41 antibody-positive patients versus 16 (15.8%) of 101 negative; in the positive group, abnormalities were generalized in 16 (43.2%), focal in seven (18.9%), multifocal in 12 (32.4%), and nonspecific in two (5.4%) patients.
Of the total group of patients, results of a complete blood count were available for 109. A low hemoglobin value (compared with the reference values for age) was found in nine of 32 antibody-positive and 11 of 77 antibody-negative patients, with no statistical difference between the two groups. Thrombocytopenia also was no more prevalent in the positive than in the negative group. Three patients in the positive group and only one in the negative group had concurrent anemia and thrombocytopenia. No patient had a history of previous thrombosis or other SLE-related features, and no patient was born of a mother with SLE.
As for AEDs, 32 positive patients were under treatment with valproate (VPA; 78% vs. 76.2% of the negative ones) and 12 with lamotrigine (LTG; 29.3% vs. 27.7% of the negative ones); other drugs such as carbamazepine (CBZ), phenobarbital (PB), clobazam (CLB), vigabatrin (VGB), ethosuximide (ESM), and felbamate (FBM) were used by fewer than five positive patients each. Detailed data are presented in Table 4. No significant differences were found between positive patients and negative ones in the type of AED used, whether alone or in association with other AEDs.
Table 4. Antiepileptic drugs (AEDs) distribution in all patients studied
Mono, monotherapy; Assoc., drug associated with other AEDs; VPA, valproate; LTG, lamotrigine; CBZ, carbamazepine; CLB, clobazam; PB, phenobarbital; VGB, vigabatrin; ESM, ethosuximide; FBM, felbamate; DZP, diazepam; No therapy, no AEDs at the moment of the blood sampling.
On the contrary, antibody positivity tended to correlate with frequency of seizures, because in patients who were antibody-positive, there were more subjects with daily seizures (more than one/day: 14 of 41 vs. 11 of 101 antibody negative; p < 0.001, χ2 with Yates correction).
The correlation between the immune system and pathogenesis of epilepsy has been the subject of many studies (43). In particular, autoimmunity has been the focus of recent work, and autoantibodies such as anti-GM1 antibodies have been reported in the sera of epilepsy patients (44–46). A high percentage of aPL-positive patients also was recently found in adult epilepsy patients (20). However, it is still not clear if these autoantibodies are only an epiphenomenon or whether they have a true pathogenetic role.
It is now widely accepted that pathogenic aPL are directed against PL-binding proteins. Among them, β2-glycoprotein I, which bears epitopes for aPL binding, has been extensively studied. These epitopes are exposed when β2GPI binds to negatively charged molecules such as cardiolipin-coated or γ–irradiated plastic plates. The importance for the determination of both aCL and anti-β2GPI antibodies has been highlighted in several reports (47–50). Elevated anti-β2GPI levels have been shown to be significantly associated with thrombosis in both the primary and secondary APS (51,52). Moreover, anti-β2GPI antibodies as detected by ELISA could have a higher specificity for thrombosis than the antibodies measured by conventional aCL ELISA, both in the primary and secondary forms of APS (53–58).
Prothrombin is an additional PL-binding protein that can be recognized by aPL-positive sera, and aPT antibodies have been reported to affect the coagulation process and to be responsible for LA activity. From a clinical point of view, aPT antibodies correlate with thrombotic events in many of the published studies, even if comparisons between series are difficult because of the technical heterogeneity of the assays used (31,32,59).
Ischemic events such as focal cerebral ischemia or ocular ischemia are the most common neurologic disorders associated with aPL; however, myelopathy, Guillain–Barré syndrome, migraine, and chorea also have been frequently reported in aPL-positive patients (60–64). A recent article showed that aPL can mediate their effects by direct interaction with neuronal tissue (29). In particular, the authors described the functional interactions of aPL with neuronal cell membranes, showing that purified IgG from a patient with aPL syndrome induced depolarization of synaptoneurosomes. Because excitatory manifestations such as epilepsy or chorea could be provoked by depolarization of synaptic membranes, a possible role for aPL in the pathogenesis of epilepsy can be postulated.
Our study showed a high prevalence of aCL, anti-β2GPI, and aPT antibodies in young patients with epilepsy. These findings agree with previous data reported in a large series of adult epilepsy patients, as well as in published smaller pediatric series and for the first time reported the presence of aPT in epilepsy patients.
In the recent article by Verrot et al. (20), 163 consecutive unselected epilepsy patients aged 14–68 years were tested for IgG and IgM aCL antibodies with ELISA, and for ANAs with immunofluorescence. IgG aCL were detected in 19% of cases versus only 3% detected in 100 control sera, whereas ANAs were found in 25% of cases. However, none of the positive tests was repeated, and the current definition of APS requires at least two consecutive positive determinations to exclude transient positive results. Moreover, in this study, contrary to ours, only β2 dependence was investigated, and not anti-β2GPI per se.
In a first report by Herranz et al. (18), a large series of 221 unselected patients with SLE had been screened for aCL and lupus anticoagulant. A statistically significant association between moderate to high titers of IgG aCL and seizures was found; additionally all 14 patients with abnormal CT or MRI had a positive test. Vascular occlusions of small cerebral vessels was postulated to be responsible for the occurrence of seizures, in at least some of the patients with SLE; confounding issues are, however, represented by the presence of various other possible comorbid factors, and by the large baseline positivity for aCL in SLE patients.
aPL antibodies have not been studied as extensively in children as in adults. In an article by Angelini et al. (15), 10 of 13 children aged 5–16 years with idiopathic cerebral ischemia were found to be positive for either lupus anticoagulant (LA) or aCL, as compared with none of LA positivity in 20 healthy controls; of 42 normal children tested in the same laboratory, none had IgG and only 5% had IgM aCL positivity. The authors suggested that it is unlikely that aPL represents an epiphenomenon for brain damage, because older adults with stroke are usually aPL-negative, despite the presence of cerebral ischemic damage. Authors from the same group recently reported persistent aPL positivity in three of 23 children with partial epileptic seizures (23). No ischemic alterations were, however, seen on either CT or MRI scans. In a control group of 40 age- and sex-matched children with other neurologic diseases, no patient was positive for LA, and none was positive for aPL. In all cases, the seizures were diagnosed as cryptogenic, unlike our series and the French one, in which all clinical diagnoses were represented.
In our series we did not investigate the presence of LA activity. However, we performed both anti-β2GPI and aPT antibody detection because it is widely accepted that anti-β2GPI and aPT antibodies can identify almost all the LA-positive samples (31,32,38,65).
It is worth noting that, in our series, four of 35 positive versus four of 82 negative patients had diffuse ischemic lesions at CT and/or MRI scan. aPL antibodies have been suggested to favor thrombus formation when a second triggering event occurs. In this regard, the antibodies we have described could have had a role as a second hit in hypoxic damage in high-risk patients. The fact that there was no female predominance in our patient group is in line with primary aPL syndrome (PAPS) patients, as opposed to SLE, in which there is a high female-to-male ratio. Moreover, a higher proportion of our aPL-positive patients had daily seizures. This phenomenon cannot be easily explained, but it could be that this finding is related to a group of patients with more severe disease. Alternatively, a role for AEDs in inducing autoantibodies cannot be ruled out, as has been described (66). However, there seems to be no relation between such drugs and antibody positivity in our series.
In conclusion, our study suggests that aCL, anti-β2GPI, and aPT can be associated with epilepsy in pediatric patients. Although a direct causal relation cannot be advocated now, we think that screening for those autoantibodies might prove useful for an early diagnosis of diseases with an autoimmune background, and could suggest alternative therapeutic approaches (i.e., anti-aggregation), in difficult cases or in patients not responsive to currently used conventional AEDs.