Original Paper Neuroimmunomodulation 2015;22:337–341 DOI: 10.1159/000371492
Received: July 28, 2014 Accepted after revision: December 5, 2014 Published online: February 17, 2015
No Overlap among Serum GAD65, NMDAR and AQP4 Antibodies in Patients with Neuromyelitis Optica Spectrum Disorders Longchang Xie a, b Youming Long a, b Ning Yang c Fulan Shan a, b Yongxiang Fan a, b Rong Zhong a, b Linzhan Wu a, b Jianrui Yin a, b Qingchun Gao a, b Gao Cong a, b a
Department of Neurology and b Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and The Ministry of Education of China, Institute of Neuroscience, Second Affiliated Hospital of Guangzhou Medical University, and c Department of Neurology, Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Abstract Objective: To evaluate whether serum glutamic acid decarboxylase (GAD), N-methyl-D-aspartate-receptor (NMDAR), and aquaporin-4 (AQP4) autoantibodies coexist in patients with neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD). Methods: Serum samples were collected from 98 patients with NMO/NMOSD. Serum GAD65, NMDAR and AQP4 antibodies were measured using a cell-based assay. Results: A total of 63 patients (64.3%) had myelitis and optic neuritis and satisfied the revised diagnostic criteria for NMO. Longitudinally extensive transverse myelitis was seen on spinal cord magnetic resonance imaging, showing continuous T2-weighted signal abnormalities in at least three vertebral segments in 26 patients (26.5%); 5 patients (5.1%) had recurrent optic neuritis, and 4 patients (4.1%) had brain syndromes with optic neuritis and myelitis. None of the 98 patients had diabetes, stiff-man syndrome, or epilepsy. All 98 patients tested positive for AQP4 antibody. No patients tested positive for GAD65 and NMDAR antibodies. Conclusions:
© 2015 S. Karger AG, Basel 1021–7401/15/0225–0337$39.50/0 E-Mail [email protected] www.karger.com/nim
In the present study, we found no simultaneous presence of serum GAD65, NMDAR and AQP4 antibodies in patients with NMO/NMOSD. © 2015 S. Karger AG, Basel
Introduction
Neuromyelitis optica (NMO) is a severe idiopathic, immune-mediated, inflammatory demyelinating and necrotizing disease, which selectively affects the optic nerves and spinal cord but typically spares the brain and follows a relapsing disease course [1–3]. The exact etiology and mechanisms of NMO are unknown. NMO lesions show a marked deposition of immunoglobulin and complement in a characteristic perivascular rosette pattern along the outer rim of thickened vessel walls [4]. NMO has been associated with many other autoimmune diseases and even more frequently with the presence of circulating autoantibodies in the absence of clinical manifestations of other diseases [5–7]. A specific IgG autoantibody selectively targeting aquaporin-4 (AQP4) has been established as a useful biomarker to distinguish NMO from multiple sclerosis (MS) [3, 8, 9]. Patients with posiDr. Gao Cong Department of Neurology Second Affiliated Hospital of Guangzhou Medical University Guangzhou, 510260 Guangdong (China) E-Mail smilegaocong @ 126.com
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Key Words Aquaporin-4 · Antibody · Glutamic acid decarboxylase · N-methyl-D-aspartate-receptor · Neuromyelitis optica
tive AQP4 antibody are usually diagnosed with NMO and its spectrum disorder (NMOSD). Glutamic acid decarboxylase (GAD) is an enzyme that catalyzes the conversion of glutamic acid from the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and is selectively expressed in GABAergic neurons and pancreatic β-cells [10]. GAD antibodies were reported to be present in patients with stiff-man syndrome, epilepsy and type 1 diabetes mellitus (T1DM) [11–14]. It has previously been shown that the T1DM-associated haplotype, HLA-B18-DR3-DQ2, associated with pancreatic autoimmune disease, is a risk factor for MS [15], suggesting that GAD may be involved in autoimmunity in MS. However, another study showed that GAD pancreatic autoimmune markers were not present among patients with MS [16]. In addition, a further study showed that 19% of 88 children with NMO had the GAD65 antibody, indicating that GAD autoimmunity may be involved in NMO [17]. Anti-N-methyl-D-aspartate-receptor (NMDAR) antibody is a biomarker associated with autoimmune limbic encephalitis. Titulaer et al. [18] identified the antiNMDAR antibody in patients with demyelinating disorder, indicating that patients with NMO and other demyelinating disorders with unusual symptoms may have anti-NMDAR encephalitis. There are few other valid data showing the frequency of sequential or coexisting antiNMDAR encephalitis in patients with the AQP4 antibody. In this study, we aimed to investigate whether serum GAD65, NMDAR and AQP4 antibodies overlap in patients with NMO/NMOSD.
Magnetic Resonance Imaging Analysis Brain and spinal magnetic resonance imaging (MRI) scans were performed on all patients using a 1.5-tesla MRI scanner. The slice thickness of the axial scans was 3–5 mm. Conventional MRI protocols were used for all patients, as follows: T1 with and without gadolinium enhancement (400/15.5 ms, TR/TE) and T2 (2,500– 3,500/100 ms, TR/TE) for spinal cord MRI, and T2 (4,600– 4,640/97.8–102 ms, TR/TE) and fluid attenuated inversion recovery (8,800/120 ms, TR/TE) for brain MRI. All MRI scans were performed prior to beginning corticosteroid treatment. No patients were receiving interferon-β or immunosuppressants at the time of MRI scanning. Serological Testing for AQP4, GAD65 and NMDAR Antibodies by Indirect Immunofluorescence Assay All serum samples were stored at −80 ° C in a laboratory of the Second Affiliated Hospital of Guangzhou Medical University. The AQP4 antibody was determined using an anti-AQP4 antibody assay on an AQP4-transfected cell line (human embryonic kidney 293 cells) from a commercial kit (Euroimmun, Luebeck, Germany). Briefly, 10 μl of serum was diluted in 90 μl of phosphate-buffered saline (PBS; 1:10). The diluted sample was added to AQP4-transfected cells placed on glass slides for 30 min at room temperature, after which the slides were rinsed 3 times with PBS. Then, fluorescein-conjugated goat antibody was incubated with the slides for 30 min. Finally, the slides were rinsed with PBS, and fluorescence was detected using a microscope. Sera were scored negative or positive by two independent assessors unaware of the clinical diagnosis. Green staining indicated cells successfully transfected with the AQP4 gene (fig. 1a). The GAD65- and NMDAR-transfected cell lines were obtained from Euroimmun. The procedures of the tests for these antibodies were similar to those described above for AQP4 antibody detection. Briefly, after incubation with serum (1: 10) for 30 min, the substrates were washed 3 times in PBS and incubated with antihuman or anti-rabbit IgG for 30 min; finally, the slides were washed in PBS. Images were captured using a Leica microscope. Typical positive NMDAR and GAD65 antibodies are shown in figure 1b and c, respectively.
Patients and Methods
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Results
A total of 63 patients (64.3%) who had myelitis and optic neuritis satisfied the revised diagnostic criteria for NMO [3]. Longitudinally extensive transverse myelitis was seen on spinal cord MRI, showing continuous T2weighted signal abnormalities in at least three vertebral segments [23] in 26 patients (26.5%); 5 patients (5.1%) had recurrent optic neuritis, and 4 patients (4.1%) had brain syndrome with optic neuritis and myelitis. None of the 98 patients had diabetes, stiff-man syndrome or epilepsy. However, 11 patients (11.2%) with hypothalamic lesions experienced confusion or decreased consciousness. All of the 98 patients tested positive for the AQP4 antibody. None of the patients tested positive for GAD65 Xie/Long/Yang/Shan/Fan/Zhong/Wu/ Yin/Gao/Cong
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Patients and Sera Sera from 98 patients were banked as part of the demyelinating diseases database in the Department of Neurology, Second Affiliated Hospital of Guangzhou Medical University, from January 2004 to April 2014. Patient samples were obtained before treatment with glucocorticoids or immunosuppressive agents. Clinical data of the patients were collected from their medical records. Informed consent was obtained from all patients for this study. Data acquired from each admission record included age, sex, medication, number of demyelinating events, clinical characteristics of each relapse, and Expanded Disability Status Scale (EDSS) score assessed at the most recent clinical examination [19]. Patients with NMO met the 2006 diagnostic criteria [20]. Patients with positive AQP4 antibodies, but who did not meet NMO criteria, were diagnosed as NMOSD, according to previous studies [21, 22].
Color version available online
and NMDAR antibodies. The demographic characteristics and other autoantibodies of the patients are presented in table 1.
Discussion
GAD65, NMDAR and AQP4 Antibodies in NMOSD
Anti-AQP4
a
Anti-NMDAR
b
Anti-GAD65
c
Fig. 1. Staining patterns of immunofluorescence in 3 types of antibodies. Green staining (color in online version only) indicates cells successfully transfected. a Anti-AQP4 pattern. b AntiNMDAR. c Anti-GAD65 pattern.
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There are few valid data showing the frequency of sequential or coexisting GAD65 and NMDAR antibodies and NMO/NMOSD. Only one report suggested the coexistence of NMDAR and AQP4 or myelin oligodendrocyte glycoprotein antibodies in 7 of 11 cases [18]. Another report identified 88 consecutive seropositive children, where 19% tested positive for the GAD65 antibody by enzyme-linked immunosorbent assay [17]. However, a negative result was found in the present study. Indeed, we did not find any overlap among AQP4, NMDAR and GAD65 antibodies. The discrepancy between the present and previous studies may have three possible causes. First, is the bias from different design methods used in the previous and present studies? Titulaer et al. [18] screened patients with NMDAR encephalitis for the AQP4 antibody, whereas we screened patients with NMOSD for the NMDAR antibody. Second, population or ethnicity may be factors. For example, AQP4 and GAD65 central nervous system autoimmunity was found in children, reflecting a special population. However, we only tested adults in the present study, whereas McKeon et al. [17] tested only children. There is little evidence showing that ethnicity influences the prevalence of GAD and NMDAR autoimmunity, but this should be examined in a wider variety of ethnic groups to confirm a lack of effect. Third, bias from different detection systems used may also affect study results, as described in a recent review [24]. In the present study, we used a highly sensitive and specific commercial kit that has been widely used in many different studies. Accordingly, the present result is likely to be valid. GAD is expressed in pancreatic β-cells and consists of two isoforms, GAD65 and GAD67, encoded by two different genes on chromosomes 10 and 2, respectively [10, 25]. Serum antibody levels of GAD65 are considered a biomarker for autoimmune diabetes and stiff-man syndrome, because they can be found in the serum of the majority of patients even before clinical onset of the disease. Subsequently, serum GAD antibodies have been reported in a large number of patients with other neurological disorders such as chronic cerebellar ataxia [26] and drug-resistant epilepsy [13, 14]. It has previously been shown that the concurrence of MS and T1DM
Table 1. Demographic characteristics and antibodies of 98 patients with NMOSD
Diagnosis
Female/male
Age at onset, years
Duration, months
AQP4, %
GAD65, %
NMDAR, %
Total (n = 98) NMO (n = 63) RLETM (n = 26) RON (n = 5) Others (n = 4)
89/9 59/4 21/5 5/0 4/0
34.5±12.5 33.8±12.8 36.0±12.3 36.0±9.4 25.0±10.8
54.8±52.5 54.6±51.2 43.1±62.2 20±15.5 50.5±45.5
100 100 100 100 100
0 0 0 0 0
0 0 0 0 0
Others: patients with AQP4 antibody without transverse myelitis or optic neuritis. RLETM = Recurrent longitudinal extensive transverse myelitis; RON = recurrent optic neuritis.
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[18]. NMDAR receptors are crucial to synaptic transmission and plasticity. Overactivity of NMDAR receptors, leading to excitotoxicity, is a proposed underlying mechanism for epilepsy, dementia and stroke [31], whereas underactivity produces emotional behavioral disturbances. Titulaer et al. [18] hypothesized that patients with antiNMDAR encephalitis may develop concurrent or separate episodes of NMO with atypical symptoms (dyskinesia, psychosis). In our study, 11 patients who had experienced confusion or decreased consciousness were included, but none had the NMDAR antibody. Their emotional behavioral disturbances may be associated with hypothalamic abnormality, reflecting no NMDAR autoimmunity involvement in our group. Previous data support that demyelinating episodes, especially NMO with AQP4 antibody, do not overlap with anti-NMDAR encephalitis [32]. Thus, whether patients with NMDAR encephalitis also have NMO/NMOSD is unclear. A major limitation of the current study is that NMDAR and GAD65 antibodies from cerebral spinal fluid, where sensitivity is higher than in serum [31], were not analyzed. Future studies testing larger patient numbers and simultaneously testing multiple biomarkers in cerebral spinal fluid are needed. However, in the present study, we found no instance of the simultaneous presence of serum GAD65, NMDAR and AQP4 antibodies in 98 patients with NMO/NMOSD. Acknowledgment This project was supported by the Guangdong Natural Science Foundation (S2013010016262).
Disclosure Statement The authors declare that no competing interests exist.
Xie/Long/Yang/Shan/Fan/Zhong/Wu/ Yin/Gao/Cong
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among a Sardinian population was related to the ancestral ‘Sardinian’ DR3* haplotype (HLA-B18-DRB1*0301DQB1 0201) – a risk factor for both disorders [15]. The question has been raised as to whether serum GAD antibodies can also be used as markers for MS. However, Bilbao et al. [16] demonstrated that GAD autoantibody frequencies are not different from the control population. Thus, there is no evidence for GAD-related autoimmunity among patients with MS. The role of GAD antibodies in other neurological diseases is thought to be in humoral immune responses to GAD that could lead to a functional impairment of GABAergic synaptic transmission [27]. Astrocytes lacking AQP4 express a reduced level of the astrocytic Na+dependent excitatory amino acid transporter 2 (EAAT2) [28], suggesting that AQP4 antibodies may alter the expression of EAAT2 in astrocyte membranes. EAAT2 accounts for approximately 90% of glutamate uptake in the central nervous system and is critical for clearing glutamate from excitatory synapses. Previous work has suggested that AQP4 antibodies contribute to pathogenesis by internalizing AQP4 and the associated glutamate transporter EAAT2, leading to glutamate excitotoxicity [29]. However, little or no internalization of AQP4 antibodies, AQP4 or EAAT2 was observed in primary astrocyte cultures, nor was glutamate uptake affected by AQP4 antibody exposure. This indicates that astrocyte endocytosis of AQP4 antibodies, AQP4 and EAAT2 is an unlikely consequence of AQP4 autoantibodies in vivo [30]. In the present study, we did not observe the GAD65 antibody in patients with AQP4 antibody status. Therefore, there is also no evidence for AQP4 antibody autoimmunity related to GAD antibodies in patients with NMO. As described in a previous report, the presence of the NMDAR antibody is observed in patients with demyelinating disorders such as acute demyelinating encephalomyelitis, myelitis or NMO, without AQP4 antibodies
References
GAD65, NMDAR and AQP4 Antibodies in NMOSD
11 De Aizpurua HJ, Wilson YM, Harrison LC: Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes. Proc Natl Acad Sci U S A 1992;89:9841–9845. 12 Solimena M, Folli F, Aparisi R, Pozza G, De Camilli P: Autoantibodies to GABA-ergic neurons and pancreatic beta cells in stiff-man syndrome. N Engl J Med 1990; 322: 1555– 1560. 13 Vulliemoz S, Vanini G, Truffert A, Chizzolini C, Seeck M: Epilepsy and cerebellar ataxia associated with anti-glutamic acid decarboxylase antibodies. J Neurol Neurosurg Psychiatry 2007;78:187–189. 14 Vulliemoz S, Vanini G, Truffert A, Chizzolini C, Seeck M: Epilepsy and cerebellar ataxia associated with anti-glutamic acid decarboxylase antibodies. J Neurol Neurosurg Psychiatry 2007;78:187–189. 15 Marrosu MG, Cocco E, Lai M, Spinicci G, Pischedda MP, Contu P: Patients with multiple sclerosis and risk of type 1 diabetes mellitus in Sardinia, Italy: a cohort study. Lancet 2002;359:1461–1465. 16 Bilbao JR, Villoslada P, Martin-Pagola A, Castano L: No evidence of pancreatic autoimmunity among patients with multiple sclerosis. Ann NY Acad Sci 2004;1037:133–137. 17 McKeon A, Lennon VA, Lotze T, et al: CNS aquaporin-4 autoimmunity in children. Neurology 2008;71:93–100. 18 Titulaer MJ, Hoftberger R, Iizuka T, et al: Overlapping demyelinating syndromes and anti-N-methyl-D-aspartate receptor encephalitis. Ann Neurol 2014;75:411–428. 19 Kurtzke JF: Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983; 33: 1444– 1452. 20 Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG: Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66:1485–1489. 21 Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG: The spectrum of neuromyelitis optica. Lancet Neurol 2007;6:805–815. 22 Sato DK, Nakashima I, Takahashi T, et al: Aquaporin-4 antibody-positive cases beyond current diagnostic criteria for NMO spectrum disorders. Neurology 2013;80:2210–2216.
23 Scott TF, Kassab SL, Pittock SJ: Neuromyelitis optica IgG status in acute partial transverse myelitis. Arch Neurol 2006;63:1398–1400. 24 Irani SR, Gelfand JM, Al-Diwani A, Vincent A: Cell-surface CNS autoantibodies: clinical relevance and emerging paradigms. Ann Neurol 2014;76:168–184. 25 Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ: Two genes encode distinct glutamate decarboxylases. Neuron 1991; 7: 91–100. 26 Honnorat J, Saiz A, Giometto B, et al: Cerebellar ataxia with anti-glutamic acid decarboxylase antibodies: study of 14 patients. Arch Neurol 2001;58:225–230. 27 Vianello M, Tavolato B, Giometto B: Glutamic acid decarboxylase autoantibodies and neurological disorders. Neurol Sci 2002; 23: 145– 151. 28 Zeng XN, Sun XL, Gao L, Fan Y, Ding JH, Hu G: Aquaporin-4 deficiency down-regulates glutamate uptake and GLT-1 expression in astrocytes. Mol Cell Neurosci 2007; 34: 34– 39. 29 Hinson SR, Roemer SF, Lucchinetti CF, et al: Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2. J Exp Med 2008;205:2473–2481. 30 Ratelade J, Bennett JL, Verkman AS: Evidence against cellular internalization in vivo of NMO-IgG, aquaporin-4, and excitatory amino acid transporter 2 in neuromyelitis optica. J Biol Chem 2011;286:45156–45164. 31 Dalmau J, Gleichman AJ, Hughes EG, et al: Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008;7:1091–1098. 32 Suh-Lailam BB, Haven TR, Copple SS, Knapp D, Jaskowski TD, Tebo AE: Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay. Clin Chim Acta 2013;421:1–6.
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1 Wingerchuk DM, Hogancamp WF, O’Brien PC, Weinshenker BG: The clinical course of neuromyelitis optica (Devic’s syndrome). Neurology 1999;53:1107–1114. 2 de Seze J, Lebrun C, Stojkovic T, Ferriby D, Chatel M, Vermersch P: Is Devic’s neuromyelitis optica a separate disease? A comparative study with multiple sclerosis. Mult Scler 2003; 9:521–525. 3 Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG: Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66:1485–1489. 4 Lucchinetti CF, Mandler RN, McGavern D, et al: A role for humoral mechanisms in the pathogenesis of Devic’s neuromyelitis optica. Brain 2002;125(part 7):1450–1461. 5 Pittock SJ, Lennon VA, de Seze J, et al: Neuromyelitis optica and non-organ-specific autoimmunity. Arch Neurol 2008;65:78–83. 6 Alves-Leon SV, Pimentel ML, Sant’Anna G, Malfetano FR, Estrada CD, Quirico-Santos T: Immune system markers of neuroinflammation in patients with clinical diagnose of neuromyelitis optica. Arq Neuropsiquiatr 2008; 66(3B):678–684. 7 Kira J: Neuromyelitis optica and opticospinal multiple sclerosis: mechanisms and pathogenesis. Pathophysiology 2011;18:69–79. 8 Lennon VA, Wingerchuk DM, Kryzer TJ, et al: A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364:2106–2112. 9 Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR: IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202:473–477. 10 Reetz A, Solimena M, Matteoli M, Folli F, Takei K, De Camilli P: GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synapticlike microvesicles suggests their role in GABA storage and secretion. EMBO J 1991;10:1275– 1284.
Received: July 28, 2014 Accepted after revision: December 5, 2014 Published online: February 17, 2015
No Overlap among Serum GAD65, NMDAR and AQP4 Antibodies in Patients with Neuromyelitis Optica Spectrum Disorders Longchang Xie a, b Youming Long a, b Ning Yang c Fulan Shan a, b Yongxiang Fan a, b Rong Zhong a, b Linzhan Wu a, b Jianrui Yin a, b Qingchun Gao a, b Gao Cong a, b a
Department of Neurology and b Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and The Ministry of Education of China, Institute of Neuroscience, Second Affiliated Hospital of Guangzhou Medical University, and c Department of Neurology, Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Abstract Objective: To evaluate whether serum glutamic acid decarboxylase (GAD), N-methyl-D-aspartate-receptor (NMDAR), and aquaporin-4 (AQP4) autoantibodies coexist in patients with neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD). Methods: Serum samples were collected from 98 patients with NMO/NMOSD. Serum GAD65, NMDAR and AQP4 antibodies were measured using a cell-based assay. Results: A total of 63 patients (64.3%) had myelitis and optic neuritis and satisfied the revised diagnostic criteria for NMO. Longitudinally extensive transverse myelitis was seen on spinal cord magnetic resonance imaging, showing continuous T2-weighted signal abnormalities in at least three vertebral segments in 26 patients (26.5%); 5 patients (5.1%) had recurrent optic neuritis, and 4 patients (4.1%) had brain syndromes with optic neuritis and myelitis. None of the 98 patients had diabetes, stiff-man syndrome, or epilepsy. All 98 patients tested positive for AQP4 antibody. No patients tested positive for GAD65 and NMDAR antibodies. Conclusions:
© 2015 S. Karger AG, Basel 1021–7401/15/0225–0337$39.50/0 E-Mail [email protected] www.karger.com/nim
In the present study, we found no simultaneous presence of serum GAD65, NMDAR and AQP4 antibodies in patients with NMO/NMOSD. © 2015 S. Karger AG, Basel
Introduction
Neuromyelitis optica (NMO) is a severe idiopathic, immune-mediated, inflammatory demyelinating and necrotizing disease, which selectively affects the optic nerves and spinal cord but typically spares the brain and follows a relapsing disease course [1–3]. The exact etiology and mechanisms of NMO are unknown. NMO lesions show a marked deposition of immunoglobulin and complement in a characteristic perivascular rosette pattern along the outer rim of thickened vessel walls [4]. NMO has been associated with many other autoimmune diseases and even more frequently with the presence of circulating autoantibodies in the absence of clinical manifestations of other diseases [5–7]. A specific IgG autoantibody selectively targeting aquaporin-4 (AQP4) has been established as a useful biomarker to distinguish NMO from multiple sclerosis (MS) [3, 8, 9]. Patients with posiDr. Gao Cong Department of Neurology Second Affiliated Hospital of Guangzhou Medical University Guangzhou, 510260 Guangdong (China) E-Mail smilegaocong @ 126.com
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Key Words Aquaporin-4 · Antibody · Glutamic acid decarboxylase · N-methyl-D-aspartate-receptor · Neuromyelitis optica
tive AQP4 antibody are usually diagnosed with NMO and its spectrum disorder (NMOSD). Glutamic acid decarboxylase (GAD) is an enzyme that catalyzes the conversion of glutamic acid from the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and is selectively expressed in GABAergic neurons and pancreatic β-cells [10]. GAD antibodies were reported to be present in patients with stiff-man syndrome, epilepsy and type 1 diabetes mellitus (T1DM) [11–14]. It has previously been shown that the T1DM-associated haplotype, HLA-B18-DR3-DQ2, associated with pancreatic autoimmune disease, is a risk factor for MS [15], suggesting that GAD may be involved in autoimmunity in MS. However, another study showed that GAD pancreatic autoimmune markers were not present among patients with MS [16]. In addition, a further study showed that 19% of 88 children with NMO had the GAD65 antibody, indicating that GAD autoimmunity may be involved in NMO [17]. Anti-N-methyl-D-aspartate-receptor (NMDAR) antibody is a biomarker associated with autoimmune limbic encephalitis. Titulaer et al. [18] identified the antiNMDAR antibody in patients with demyelinating disorder, indicating that patients with NMO and other demyelinating disorders with unusual symptoms may have anti-NMDAR encephalitis. There are few other valid data showing the frequency of sequential or coexisting antiNMDAR encephalitis in patients with the AQP4 antibody. In this study, we aimed to investigate whether serum GAD65, NMDAR and AQP4 antibodies overlap in patients with NMO/NMOSD.
Magnetic Resonance Imaging Analysis Brain and spinal magnetic resonance imaging (MRI) scans were performed on all patients using a 1.5-tesla MRI scanner. The slice thickness of the axial scans was 3–5 mm. Conventional MRI protocols were used for all patients, as follows: T1 with and without gadolinium enhancement (400/15.5 ms, TR/TE) and T2 (2,500– 3,500/100 ms, TR/TE) for spinal cord MRI, and T2 (4,600– 4,640/97.8–102 ms, TR/TE) and fluid attenuated inversion recovery (8,800/120 ms, TR/TE) for brain MRI. All MRI scans were performed prior to beginning corticosteroid treatment. No patients were receiving interferon-β or immunosuppressants at the time of MRI scanning. Serological Testing for AQP4, GAD65 and NMDAR Antibodies by Indirect Immunofluorescence Assay All serum samples were stored at −80 ° C in a laboratory of the Second Affiliated Hospital of Guangzhou Medical University. The AQP4 antibody was determined using an anti-AQP4 antibody assay on an AQP4-transfected cell line (human embryonic kidney 293 cells) from a commercial kit (Euroimmun, Luebeck, Germany). Briefly, 10 μl of serum was diluted in 90 μl of phosphate-buffered saline (PBS; 1:10). The diluted sample was added to AQP4-transfected cells placed on glass slides for 30 min at room temperature, after which the slides were rinsed 3 times with PBS. Then, fluorescein-conjugated goat antibody was incubated with the slides for 30 min. Finally, the slides were rinsed with PBS, and fluorescence was detected using a microscope. Sera were scored negative or positive by two independent assessors unaware of the clinical diagnosis. Green staining indicated cells successfully transfected with the AQP4 gene (fig. 1a). The GAD65- and NMDAR-transfected cell lines were obtained from Euroimmun. The procedures of the tests for these antibodies were similar to those described above for AQP4 antibody detection. Briefly, after incubation with serum (1: 10) for 30 min, the substrates were washed 3 times in PBS and incubated with antihuman or anti-rabbit IgG for 30 min; finally, the slides were washed in PBS. Images were captured using a Leica microscope. Typical positive NMDAR and GAD65 antibodies are shown in figure 1b and c, respectively.
Patients and Methods
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Neuroimmunomodulation 2015;22:337–341 DOI: 10.1159/000371492
Results
A total of 63 patients (64.3%) who had myelitis and optic neuritis satisfied the revised diagnostic criteria for NMO [3]. Longitudinally extensive transverse myelitis was seen on spinal cord MRI, showing continuous T2weighted signal abnormalities in at least three vertebral segments [23] in 26 patients (26.5%); 5 patients (5.1%) had recurrent optic neuritis, and 4 patients (4.1%) had brain syndrome with optic neuritis and myelitis. None of the 98 patients had diabetes, stiff-man syndrome or epilepsy. However, 11 patients (11.2%) with hypothalamic lesions experienced confusion or decreased consciousness. All of the 98 patients tested positive for the AQP4 antibody. None of the patients tested positive for GAD65 Xie/Long/Yang/Shan/Fan/Zhong/Wu/ Yin/Gao/Cong
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Patients and Sera Sera from 98 patients were banked as part of the demyelinating diseases database in the Department of Neurology, Second Affiliated Hospital of Guangzhou Medical University, from January 2004 to April 2014. Patient samples were obtained before treatment with glucocorticoids or immunosuppressive agents. Clinical data of the patients were collected from their medical records. Informed consent was obtained from all patients for this study. Data acquired from each admission record included age, sex, medication, number of demyelinating events, clinical characteristics of each relapse, and Expanded Disability Status Scale (EDSS) score assessed at the most recent clinical examination [19]. Patients with NMO met the 2006 diagnostic criteria [20]. Patients with positive AQP4 antibodies, but who did not meet NMO criteria, were diagnosed as NMOSD, according to previous studies [21, 22].
Color version available online
and NMDAR antibodies. The demographic characteristics and other autoantibodies of the patients are presented in table 1.
Discussion
GAD65, NMDAR and AQP4 Antibodies in NMOSD
Anti-AQP4
a
Anti-NMDAR
b
Anti-GAD65
c
Fig. 1. Staining patterns of immunofluorescence in 3 types of antibodies. Green staining (color in online version only) indicates cells successfully transfected. a Anti-AQP4 pattern. b AntiNMDAR. c Anti-GAD65 pattern.
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There are few valid data showing the frequency of sequential or coexisting GAD65 and NMDAR antibodies and NMO/NMOSD. Only one report suggested the coexistence of NMDAR and AQP4 or myelin oligodendrocyte glycoprotein antibodies in 7 of 11 cases [18]. Another report identified 88 consecutive seropositive children, where 19% tested positive for the GAD65 antibody by enzyme-linked immunosorbent assay [17]. However, a negative result was found in the present study. Indeed, we did not find any overlap among AQP4, NMDAR and GAD65 antibodies. The discrepancy between the present and previous studies may have three possible causes. First, is the bias from different design methods used in the previous and present studies? Titulaer et al. [18] screened patients with NMDAR encephalitis for the AQP4 antibody, whereas we screened patients with NMOSD for the NMDAR antibody. Second, population or ethnicity may be factors. For example, AQP4 and GAD65 central nervous system autoimmunity was found in children, reflecting a special population. However, we only tested adults in the present study, whereas McKeon et al. [17] tested only children. There is little evidence showing that ethnicity influences the prevalence of GAD and NMDAR autoimmunity, but this should be examined in a wider variety of ethnic groups to confirm a lack of effect. Third, bias from different detection systems used may also affect study results, as described in a recent review [24]. In the present study, we used a highly sensitive and specific commercial kit that has been widely used in many different studies. Accordingly, the present result is likely to be valid. GAD is expressed in pancreatic β-cells and consists of two isoforms, GAD65 and GAD67, encoded by two different genes on chromosomes 10 and 2, respectively [10, 25]. Serum antibody levels of GAD65 are considered a biomarker for autoimmune diabetes and stiff-man syndrome, because they can be found in the serum of the majority of patients even before clinical onset of the disease. Subsequently, serum GAD antibodies have been reported in a large number of patients with other neurological disorders such as chronic cerebellar ataxia [26] and drug-resistant epilepsy [13, 14]. It has previously been shown that the concurrence of MS and T1DM
Table 1. Demographic characteristics and antibodies of 98 patients with NMOSD
Diagnosis
Female/male
Age at onset, years
Duration, months
AQP4, %
GAD65, %
NMDAR, %
Total (n = 98) NMO (n = 63) RLETM (n = 26) RON (n = 5) Others (n = 4)
89/9 59/4 21/5 5/0 4/0
34.5±12.5 33.8±12.8 36.0±12.3 36.0±9.4 25.0±10.8
54.8±52.5 54.6±51.2 43.1±62.2 20±15.5 50.5±45.5
100 100 100 100 100
0 0 0 0 0
0 0 0 0 0
Others: patients with AQP4 antibody without transverse myelitis or optic neuritis. RLETM = Recurrent longitudinal extensive transverse myelitis; RON = recurrent optic neuritis.
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[18]. NMDAR receptors are crucial to synaptic transmission and plasticity. Overactivity of NMDAR receptors, leading to excitotoxicity, is a proposed underlying mechanism for epilepsy, dementia and stroke [31], whereas underactivity produces emotional behavioral disturbances. Titulaer et al. [18] hypothesized that patients with antiNMDAR encephalitis may develop concurrent or separate episodes of NMO with atypical symptoms (dyskinesia, psychosis). In our study, 11 patients who had experienced confusion or decreased consciousness were included, but none had the NMDAR antibody. Their emotional behavioral disturbances may be associated with hypothalamic abnormality, reflecting no NMDAR autoimmunity involvement in our group. Previous data support that demyelinating episodes, especially NMO with AQP4 antibody, do not overlap with anti-NMDAR encephalitis [32]. Thus, whether patients with NMDAR encephalitis also have NMO/NMOSD is unclear. A major limitation of the current study is that NMDAR and GAD65 antibodies from cerebral spinal fluid, where sensitivity is higher than in serum [31], were not analyzed. Future studies testing larger patient numbers and simultaneously testing multiple biomarkers in cerebral spinal fluid are needed. However, in the present study, we found no instance of the simultaneous presence of serum GAD65, NMDAR and AQP4 antibodies in 98 patients with NMO/NMOSD. Acknowledgment This project was supported by the Guangdong Natural Science Foundation (S2013010016262).
Disclosure Statement The authors declare that no competing interests exist.
Xie/Long/Yang/Shan/Fan/Zhong/Wu/ Yin/Gao/Cong
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among a Sardinian population was related to the ancestral ‘Sardinian’ DR3* haplotype (HLA-B18-DRB1*0301DQB1 0201) – a risk factor for both disorders [15]. The question has been raised as to whether serum GAD antibodies can also be used as markers for MS. However, Bilbao et al. [16] demonstrated that GAD autoantibody frequencies are not different from the control population. Thus, there is no evidence for GAD-related autoimmunity among patients with MS. The role of GAD antibodies in other neurological diseases is thought to be in humoral immune responses to GAD that could lead to a functional impairment of GABAergic synaptic transmission [27]. Astrocytes lacking AQP4 express a reduced level of the astrocytic Na+dependent excitatory amino acid transporter 2 (EAAT2) [28], suggesting that AQP4 antibodies may alter the expression of EAAT2 in astrocyte membranes. EAAT2 accounts for approximately 90% of glutamate uptake in the central nervous system and is critical for clearing glutamate from excitatory synapses. Previous work has suggested that AQP4 antibodies contribute to pathogenesis by internalizing AQP4 and the associated glutamate transporter EAAT2, leading to glutamate excitotoxicity [29]. However, little or no internalization of AQP4 antibodies, AQP4 or EAAT2 was observed in primary astrocyte cultures, nor was glutamate uptake affected by AQP4 antibody exposure. This indicates that astrocyte endocytosis of AQP4 antibodies, AQP4 and EAAT2 is an unlikely consequence of AQP4 autoantibodies in vivo [30]. In the present study, we did not observe the GAD65 antibody in patients with AQP4 antibody status. Therefore, there is also no evidence for AQP4 antibody autoimmunity related to GAD antibodies in patients with NMO. As described in a previous report, the presence of the NMDAR antibody is observed in patients with demyelinating disorders such as acute demyelinating encephalomyelitis, myelitis or NMO, without AQP4 antibodies
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