Immune-mediated epilepsy with GAD65 antibodies

Journal Pre-proof Immune-mediated epilepsy with GAD65 antibodies

Xinxin Li, Qi Guo, Zhaoshi Zheng, Xiaoshuang Wang, Songyan Liu PII:

S0165-5728(20)30027-8

DOI:

https://doi.org/10.1016/j.jneuroim.2020.577189

Reference:

JNI 577189

To appear in:

Journal of Neuroimmunology

Received date:

11 January 2020

Revised date:

11 February 2020

Accepted date:

11 February 2020

Please cite this article as: X. Li, Q. Guo, Z. Zheng, et al., Immune-mediated epilepsy with GAD65 antibodies, Journal of Neuroimmunology (2019), https://doi.org/10.1016/ j.jneuroim.2020.577189

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© 2019 Published by Elsevier.

Journal Pre-proof Immune-mediated epilepsy with GAD65 antibodies

Xinxin Lia, Qi Guoa, Zhaoshi Zhenga, Xiaoshuang Wanga, Songyan Liub,* a

Department of Neurology, China–Japan Union Hospital of Jilin University,

Changchun, Jilin Province, China b

Department of Neurology, China–Japan Union Hospital of Jilin University, No 126,

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Corresponding author.

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*

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Xian tai Street, Changchun, 130000 Jilin Province, China.

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Abstract

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Anti-GAD65 antibodies have been identified in both acute/subacute seizures (limbic encephalitis and extralimbic encephalitis) and chronic isolated epilepsy. The evidence of

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high serum titers and intrathecal synthesis play a fundamental role in diagnosis but poorly

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correlate with disease severity or response to therapies. It remains controversial whether

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anti-GAD65 Abs are the pathogenic entity or only serve as a surrogate marker for autoimmune disorders mediated by cytotoxic T cells. Unlike other immune-mediated epilepsy, although multiple combinations of therapeutics are used, the efficacy and prognosis of patients with GAD65-epilepsy patients are poor. Besides, GAD65-epilepsy is more prone to relapse and potentially evolve into a more widespread CNS inflammatory disorder. This article reviews the recent advances of GAD65-epilepsy, focusing on the diagnosis, epidemiology, pathophysiology, clinical features, and treatment, to better promote the recognition and provide proper therapy for this condition. 1.Introduction

Journal Pre-proof Glutamic acid decarboxylase (GAD) is a key enzyme in the dynamic regulation of neural network excitability that converts glutamate into Gamma-aminobutyric acid (GABA). There are two isoforms of this enzyme: GAD65 and GAD67, which are widely distributed within the central nervous system, pancreas, and other organs[1, 2] . In patients with neurological symptoms, antibodies can be detected against GAD65, but also less frequently against GAD67. Although the significance of anti-GAD65 antibodies(anti-GAD65 Abs) has been a

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focus of debate, several neurological phenotypes of anti-GAD65 associated neurological

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disease have been described, such as stiff-person syndrome (SPS), cerebellar ataxia(CA),

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ocular movement disorder and myelitis[3-6] .Lately, anti-GAD65 Abs were found in patients with seizures in the context of encephalitis, and in patients with chronic epileptic

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syndromes without clinical or MRI evidence of active CNS inflammation [7-9]. In some cases,

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seizures may be the main or the only clinical presentation in encephalitis associated with

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anti-GAD65 Abs; thus, “subacute” encephalitis and “chronic” epilepsy seem to represent

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a continuum of a rare neurological syndrome [10] . To make consistency in terminology, we collectively refer to these diseases as "immune-mediated epilepsy with anti-GAD65 Abs " (GAD65-epilepsy) [11,

12] .

This article reviews the recent advances of GAD65-epilepsy,

focusing on the diagnosis, epidemiology, pathophysiology, clinical features, and treatment, to better promote the recognition and provide proper therapy for this condition. 2.Diagnosis The diagnosis of GAD65-epilepsy is based on clinical grounds plus the presence of high anti-GAD65 Abs titers or the detection of intrathecal synthesis (IS) in CSF. Currently, several recognized high anti-GAD65 Abs levels are defined as follows: 1) usually> 20

Journal Pre-proof nmol/L or >2000 U/mL when determined by radioimmunoassay(RIA);2)> 20 nmol/L or >1000 IU/mL by Enzyme-linked immunosorbent assay (ELISA);3) screened by immunohistochemistry(IHC) and confirm the positive cases by RIA or Western Blot (WB)[10,

12-17] .

High anti-GAD65 Abs titers have high specificity for neurological

autoimmunity, and only 0.8% of patients with T1DM detected anti-GAD65 Abs titers above 2000 U/mL [13] . 20% of patients with Abs were only present in serum, and the serum Abs

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titers are often higher than CSF [10, 18] . However, in some cases, the serum Ab titers might

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be low, but still show clear evidence of IS in CSF. Therefore, Clinically, patients with highly

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suspected GAD65-epilepsy should be simultaneously screened for the titer of anti-GAD65 Abs in serum and CSF. The IS index of anti-GAD65 Abs was calculated using the values

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obtained by immunohistochemistry, based on the following formula: [CSF anti-GAD65

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Abs titer/serum anti-GAD65 Abs titer]/ [CSF albumin/serum albumin], Values higher than

19] .IS

suggests active and ongoing CNS inflammation that could identify patients

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IgG[13,

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the IgG index, particularly higher than 1, are strong indicators for IS of antibody-specific

who might be warranted an immunosuppressive therapy [1, 10] . 3.Epidemiology

Up to 80% of type I diabetes(T1DM) have low-positive anti-GAD65 Abs (< 100 U/mL); furthermore, such low-positive antibodies may have been detected in 1 to 2% of healthy people and are relatively non-specific

[20, 21] .

The true incidence of GAD65-epilepsy

remains unknown. In the existing studies, due to the method of determination and the levels of antibodies considered relevant have been variable, discrepant results do exist , and most studies are limited by lack of CSF data. Recently, a population-based study from

Journal Pre-proof Olmsted County, USA, estimated the prevalence of autoimmune encephalitis to be13.7/100,000 people，of which anti-GAD65 Abs was found to be one of

the most

common autoantibodies, with a prevalence of 1.9/100,000 [22] .A recent hospital-based prospective study reported that 8% of new-onset or established epilepsy of unknown etiology had high titers of anti-GAD65 Abs [7] .Other studies reported that high anti-GAD65 Abs titers have been detected in 2.1 to 5.4% of adult-onset focal epilepsy(more frequently

research on children, high anti-GAD65 Abs titers have been detected in 0.5% to

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23] .Less

[10, 14, 18,

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exhibiting drug-resistant seizures) and 6.5%-17% of limbic encephalitis (LE)

encephalopathies

[24-26]. Moreover,

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0.8% in pediatric epilepsy, and 2% in pediatric patients with cryptogenic forms of epileptic anti-GAD65 Abs were detected 8.7% in pediatric

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epileptic patients with an undetermined etiology and up to 35.3% in patients with

28] .

However, the diagnostic value of these low anti-GAD65 Abs titers to

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levels[27,

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encephalitis and childhood status epilepticus, but none of them reached the high titer

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neurological disease remains unclear. 4.Pathophysiology

4.1Structural and functional of the GAD The neuroendocrine enzyme GAD catalyzes the synthesis of Gamma-aminobutyric acid (GABA), which is the prime inhibitory neurotransmitter that is necessary to the control of synaptic excitation/inhibition and neural oscillation[29]. GAD involves two isoforms that catalyze GABA synthesis named according to their respective molecular weights: GAD65 and GAD67[30] . The two isoforms have the same fold and general overall sequence similarity, each having an amino (N)-terminal domain, a middle PLP-binding domain

Journal Pre-proof containing the active catalytic site of the enzyme, and a carboxy (C) -terminal domain, with the middle and C-terminal domains having 74% identity, 25% identity for the N-terminal domain[4] . GAD67 is scattered all over the cytoplasm, is an active element and constantly active to produce basal levels of GABA, whereas GAD65 is mainly located in synaptic vesicles, is temporarily activated, undergoes auto-inactivation during enzyme activity and occurs in the cell primarily as apoenzyme, providing for a pulse in production under

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circumstances that demand a rapid surge of GABA synthesis and release [30,

31] .The

inhibitory neurotransmission

subtype-specific

knockdown

animals

at

the

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subsequent

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activity and expression of the two isoforms are highly correlated with GABA levels and

have

synapses[32] .GAD67

shown

that

the

reduced

interneuron inhibitory

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neurotransmission caused by GAD67 deficiency results in behavioral changes related to

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anxiety and schizophrenia[33] .In GAD65 knockout mice, the level of GABA is a greater

only the GAD65 functions as an autoantigen, the GAD67 is rarely

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35] .However,

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than 50% decrease, and the mice appear to be more vulnerable to stress and seizures [34,

autoantigenic in isolation[36,

37] .

Comparison of GAD65 and GAD67 reveals that the

binding site of GAD65 for antibodies displays greater polarity and more negatively charged amino acid residues on its surface, and the C-terminal and catalytic loop residues of GAD65 display more flexibility and mobility, which may enhance the propensities of antigenicity[4, 30, 38]. 4.2 Epitope features of Anti-GAD65 Abs Anti-GAD65 Abs autoimmunity is associated with a variety of syndromes, from T1DM to neurological diseases such as SPS, CA, as well as limbic encephalitis (LE) and epilepsy.

Journal Pre-proof Immunodominant epitope-specific recognition is considered to be one of the causes of different clinical symptoms. Studies disclosed that antibodies epitopes were tightly clustered in the region surrounding the C-terminal flexible region, and segregated into two distinct C-terminal clusters (b78 and 96.11) located to opposite faces of the C-terminal domain[4, 39] . Anti-GAD65 Abs from patients with neurological disorders have been shown to preferentially recognize the b78 epitope specificity, which tended to be enzyme

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inhibitory, while that in T1DM patients recognize more commonly the b96.11 epitope

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specificity, which tended to be non-enzyme inhibitory [40-42] . Antibodies against the b78

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epitope have demonstrated to interferes with the association of GAD65 with the cytosolic face of GABA-containing synaptic vesicles, inhibit the enzymatic activity of GAD65.

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Besides, cerebellar slice experiments suggested a gradual but sustained inhibitory effect

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of the b78 epitope on the GABAergic synaptic transmission between basket cells and

In neurological disorders linked to anti-GAD65 Abs, researches on specific epitopes

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44].

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Purkinje cells. These effects were not observed with Abs against the b96.11 epitope[1, 41, 43,

recognition was contradictory. Vianello et al.

[45] used

immunofluorescence on cultured

hippocampal neurons, and staining patterns differed among patients with SPS, CA，and epilepsy-related to anti-GAD65 Abs; the tentative suggestion was that differences in GAD65 epitope recognition differentiated clinical phenotypes. But in two other studies of GAD65 epitope mapping, no differences in specific epitopes recognized was found among various symptoms, and only reported that the serum of patients with LE was more likely to react with the N-terminal domain, whereas epileptic patients showed more reactivity against the C-terminal domain, but this difference of epitope recognition was insignificant

Journal Pre-proof and not observed in the CSF [36,

46] .

A recent study showed that SPS patients could

recognize a linear epitope at the N-terminal region of GAD65 ，preferably, and inhibited GAD65 enzyme activity better compared to epilepsy patients

[47] ,

but these are not

sufficient, and further specific identification studies of the epitope of GAD65 are necessary. 4.3 Do Anti-GAD65 Abs Play a Pathogenic Role in GAD65-epilepsy?

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It has been postulated that the humoral immune response to anti-GAD65 Abs could

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result in reduced amounts of presynaptic available GABA content through reducing GABA

[41, 48-51] .

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synthesis in nerve terminals and interfering with exocytosis of GABA in synaptic vesicles In an MRI-spectroscopy study, low cortical GABA levels were reported in epileptic

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patients with high levels of serum anti-GAD65 Abs

[52] .

Nevertheless, it remains

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controversial that anti-GAD65 Abs are the pathogenic entity of GAD65-epilepsy. Firstly,

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the results of studies on the direct pathogenic influence of anti-GAD65 Abs affecting

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inhibitory synaptic transmission in central GABAergic pathways are inconsistent . Increased spontaneous activity of a network of hippocampal neurons in culture caused by registering on cultured hippocampal neurons following application of serum from epileptic patients with anti-GAD65 Abs while no effect was noted using sera from negative controls [53] .However,

stereotactic injection of human CSF containing anti-GAD65 Abs into the

hippocampus of rats in vivo did not alter evoked and spontaneous GABAergic synaptic transmission intact in the recent two studies[54,

55] .

Secondly, GAD65 is cytosolic and,

therefore, not readily accessible for circulating antibodies. It generally acts as a biomarker for cytotoxic T-cell mediated neuronal damage

[56] .

Nowadays, the effector functions of T

Journal Pre-proof cells in GAD65-epilepsy have attracted attention. In pathological studies of LE and unilateral TLE who underwent amygdalohippocampectomy, patchy inflammation by T cells was observed in the hippocampi. Instead, immunoglobulin and complement deposition was completely absent

[57-59] .

Experimental studies have shown that cytotoxic

t-lymphocyte attack on neurons may lead to perforin-dependent electrical silencing, thereby causing the cluster of differentiation CD8+ T cells to play a major role in neuronal

attacks

against

GAD65-expressing

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neurons[60] . Thus, these nonlethal cytotoxic

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damage in CNS inflammation, but this damage does not necessarily cause the death of

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GABAergic interneurons and ensuing cellular silence/dysfunction could lead to chronic epilepsy[12, 57].Thirdly, if anti-GAD65 Abs are pathogenic, they need to be internalized and

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reach the intracellular targets. Some experiments have confirmed the internalization of

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human monoclonal anti-GAD65Ab b78 in cultured cells or rat cerebral cortex, and have

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observed b78 in CA1 interneurons and Purkinje neurons in the medial septum/diagonal

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band and ipsilateral interpositus nucleus [41, 50, 61, 62] . However, it was not confirmed in a study involving patients with LE and epilepsy [46] .Furthermore, GAD65 can become membrane-associated or anchored to synaptic vesicles through protein complex formation with the heat shock protein 70 families

[63] .

Thus, GAD65 could be transiently

exposed on the cell surface during exocytosis from GABAergic neurons. Alternatively, the antibodies may be endocytosed and transported internally to their cytoplasmic antigen , allowing a pathogenic antibody-antigen interaction to occur[64-66] . Moreover, anti-GAD65 Abs may coexist with additional cell-surface antibody, such as anti-NMDA-R, anti-VGKC, anti-GABAA-R, or GABAB-R[67-6 9] .These antibodies might alter neuronal function by

Journal Pre-proof receptor competition and internalization and are directly pathogenic to cells. At this time, anti-GAD65 Abs might rather be a bystander effect generated by blood-brain barrier permeability and widespread neuronal destruction [68] . 5.Clinical Features GAD65-epilepsy may be found in two different settings: acute/subacute onset of seizures or chronic epilepsy, and the clinical manifestations are heterogeneous with a

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wide clinical spectrum ranging from mild non-pharmacoresistant epilepsy to refractory

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TLE, LE, and also extralimbic encephalitis (ELE). Most cases were young adult patients,

males have a slight predominance [27, 70] .

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5.1chronic epilepsy

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and women were more frequently affected than men; however, in the pediatric population,

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Historically, Giometto was the first to describe the presence of anti-GAD65 Abs in

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patients with chronic epilepsy in 1998[71] .Most patients were focal seizures with clinical

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localization of the seizure focus to the temporal lobe, and onset in adult life with no history of antecedent cerebral insults and no epileptogenic lesions identified on MRI scanning using appropriate imaging protocols[70].The most common focal seizures were psychic, followed by somatosensorial, motor, and visual symptoms

[72] .Moreover,

recent studies

reported that 9% of to15%of patients with GAD65 epilepsy presented music-induced reflex seizures (MRS), it seems to be a distinctive seizure type of GAD65 epilepsy [72, 73] . Also, some patients experienced different seizure types during disease evolution. 75% of patients found no epileptogenic lesions identified on the first MRI scanning [72] . During the follow-up, the most common imaging abnormality was disproportionate cerebral or

Journal Pre-proof cerebellar volume loss for age. Although a minority of patients had some degree of hippocampal involvement, hippocampal sclerosis (HS) seemed to be not a dominant feature[11,

74] .In

the FDG-PET study, most patients presented hypometabolism in MTLE

and insular structures, and insular involvement could be an important diagnostic clue for GAD65-epilepsy, especially in patients with MTLE epilepsy of unknown origin [72] . 5.2 limbic encephalitis (LE) and extralimbic encephalitis (ELE)

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LE is subacute onset with progression in less than 3 months，defined by pathological

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temporal structural abnormalities on neuroimaging, temporal lobe seizures, and in part, 76] .

Sub-forms of LE is

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rapidly progressive memory and affective disturbances[75,

differentiated based on the underlying antigen. One form of LE based on neuronal

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cell-surface antibodies such as voltage-gated potassium channel (VGKC) complex Abs

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(currently known to react with LGI-1 and CASPR-2 antigens), and the other form of LE

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with antibodies targeting intracellular antigens like GAD65 [77] .The main clinical

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manifestation with LE associated with anti-GAD65 Abs are epilepsy and memory impairment, and psychiatric disorders, orientation, executive functions, and language disorders were also possible[8] .But a “craniofacial dystonia or orofacial dyskinesia ” pattern, as described in VGKC or NMDAR antibodies receptor encephalitis, is lacking in anti-GAD65 Abs-related LE[8] . Compared to patients with LE associated with anti-VGKC complex Abs, those with anti-GAD65 Abs were younger, present first with seizures rather than cognitive symptoms, had oligoclonal bands in the CSF, along with intrathecal synthesis, and showed an unremitting chronic disease course with antibodies persistence[18,

78, 79].Rare

cases may present with prominent dysautonomia, severe

Journal Pre-proof neuropsychological impairments，dementia ,ictal asystole, and may have drug-resistant TLE for several years prior to diagnosis

[80-84] .

Cases involving mixed LE and ELE

subtypes have also been described, but the seizure features were mesial temporal despite ELE involvement[85] .Isolated ELE subtype is uncommon, and only a few have been reported. ELE subtype comprises, clinically and radiographically, a more heterogeneous syndrome, varying according to the location and extent of ELE

[85-88] .

In the setting of LE, brain MRI usually shows MRI T2-weighted

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status epilepticus

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involvement, some patients developed non-convulsive status epilepticus and convulsive

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hyperintensity and "swelling" in mesial temporal structure (bilaterally in most cases), and automated mesiotemporal volumetry only displays amygdala enlargement[79, 89] . Follow up

and atrophy[59] . The

brain MRI of

ELE present

with

hyperintense

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sclerosis

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MRI in patients with prolonged and frequent seizures may demonstrate hippocampal

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cortical/subcortical lesions in T2W/FLAIR MRI sequences, progressive changes

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predominantly affecting the frontal lobes, the occipito-parietal region with some extension into the temporal lobes and other cortical regions, and usually without contrast enhancement[86,

87] .

FDG-PET may show hypermetabolism corresponding to the MRI

lesions, and DTI may show widespread white matter damage already at relatively early disease stages[90, 91] . 5.3Co-existing autoimmune antibodies and diseases Patients with GAD65-epilepsy frequently coexist one or more systemic autoimmune disorders, including diabetes ，thyroiditis，psoriasis, celiac disease, vitiligo, common variable immune deficiency, and others[8] . In addition, some patients are more likely to

Journal Pre-proof have serological evidence of multiple autoantibodies, but without any clinical signs of autoimmune

diseases,

such

antiphospholipid antibodies[10,

as 92] .

anti-TPO,

anti-gliadin,

antinuclear

antibody,

In Gagnon's report, almost half of cases have

co-existing systemic autoimmune conditions, mainly T1DM, followed by autoimmune thyroiditis[8]. These systemic autoimmune conditions and high anti-GAD65 Abs levels can precede the clinical manifestations of GAD65-epilepsy for several years[93] .Furthermore, it

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is necessary to screen and address endocrinologic abnormalities, particularly in patients

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with GAD65-epilepsy that are less responsive to immunosuppressive therapy. One case

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reported that exogenous testosterone replacement had played an important role in improving the seizures in a patient with GAD65-epilepsy and low testosterone [94] . Rare

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cases have reported the co-existence of anti-GAD65 Abs and cell-surface antibodies, and

example,

multifocal

asynchronous

sub-cortical/cortical

hyperintensities

are

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For

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this co-existence may result in the variability seen in the clinical and radiographic features.

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concerning for co-existing anti-GABAA-R antibodies[67] . Also, GAD65-epilepsy may co-exist with visual disturbances or movement disorder when anti-GlyR antibodies are present, or warning a paraneoplastic and refractory disease in the presence of anti-GABAB-R antibodies[69, 95, 96] . Nevertheless, in some cases, the titer of anti-GAD65 Abs was present at a much lower than that in neurologic syndromes and was not related to disease activity[68] . Moreover, some patients with GAD65-epilepsy may evolve into a more widespread CNS inflammatory disorder at some point in their illness, such as SPS, CA, optic neuritis and transverse myelitis, these syndromes may represent a continuum of anti-GAD65 Abs -associated CNS disease [70, 97-99] .

Journal Pre-proof 5.4Paraneoplastic associations Previous studies have suggested that GAD65-epilepsy often associated with non-paraneoplastic forms

[12] ,

little is known of anti-GAD65 Abs in the paraneoplastic

context. However, anti-GAD65 Abs have been detected in patients with lung carcinomas (small-cell and non-small cell), thymoma, breast cancer, and testicular seminoma [100-102] . Although this antibody has less sensitivity and specificity for the presence of malignancy,

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and are not included as a “classic” paraneoplastic antibody, anti-GAD65 Abs are already

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included in commercially available paraneoplastic line-blots[103] .Paraneoplastic etiology in

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anti-GAD65 Abs associated isolate epilepsy is extremely rare [104] . The probability of malignancy with anti-GAD65 Abs increases if the presentation matches a classic

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paraneoplastic syndrome or typical of LE with rapid disease progression[101] . Furthermore,

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it has been shown that the probability of an underlying malignancy was a 7-fold increased

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risk in patients with anti-GAD65 Abs and co-existing neuronal cell-surface antibodies,

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particularly in male patients older than 50 years of age with concomitant anti-GABAB receptor Abs[95,

101, 102] .In

these cases, appropriate and targeted malignancy screening

should be performed. paraneoplastic antibodies may be earlier than any evidence of primary malignancy, so continued, regular malignancy screening is necessary , it is recommended to be at least once every six months for four years [105, 106] . 6.Treatment The treatment of patients with GAD65-epilepsy is comprised of symptomatic therapy (including antiepileptic drugs), immunotherapy, and surgical therapies. Most patients received multiple therapeutic interventions in varying combinations, but the response and

Journal Pre-proof prognosis were poor. Moreover, current treatment recommendations lack randomized controlled trials and are mainly based on case series and clinical experience . Recently, a special study emphasis on treatment timing and the relationship between immunotherapy and anti-epileptic drugs GAD65-epilepsy

into three main

[107] .They

stages:

In

the

divided the clinical course of first

stage, acute reversible

immune-activation causes the first seizure, there is no permanent brain damage, and no

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visible brain MRI changes [108] . The main focus of treatment should be on immunotherapy,

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and early initiation of immunotherapy can prevent from developing refractory epilepsy

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even provides complete seizure freedom. In the second stage, there is already subtle irreversible brain damage, which causes MRI abnormalities and refractory epilepsy[58] .

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Immunotherapy is still effective and can resolve edema and abnormal signal changes of

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MRI. However, after that, focus management should shift to refractory epilepsy. In the

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third stage, there was post-inflammatory astrogliosis causing secondary hippocampal

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sclerosis or to more diffuse brain damage, and it was often considered the underlying “structural” cause of the seizures. Immunotherapy seems to be ineffective, and treatment should focus on seizure control

[91, 109] .

However, for patients with pharmacoresistant

epilepsy without an acute onset, the mentioned above three stages do not seem to explain the clinical course, and the routine use of immunotherapy has not been efficacious [70, 110] .

6.1Immunotherapy regimens The immunotherapy protocol may be subdivided into first-line (immunomodulatory treatment) and second-line therapies (immunosuppressive treatment).First-line therapies

Journal Pre-proof include high-dose intravenous methylprednisolone (IVMP), intravenous immune globulin (IVIG), or plasma exchange (PLEX) [111] . High-dose IVMP (1g per day for 5 days, followed by monthly pulsatile treatment or oral prednisone) were the most frequent applied first-line therapies, with 45% of patients achieving seizure response (50% reduction) . However, relevant side effects were observed in 50% of the patients. IVIG (0.4 g/kg per day for 5 consecutive days) and immunoadsorption were less effective in seizure response but with In some cases, PLEX (5–7 cycles; 1

f

112] .

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better tolerability than corticosteroids [11,

[113, 114] .

For patients with improvement, gradual

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antibody titers and improve seizures

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exchange every other day spread over 10–14 days)can effectively diminish anti-GAD65

reduction in dosing may be required, including a slow taper of oral prednisone, or a

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gradual lengthening of the intervals between intravenous treatments [102,

111] .

In some

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scenarios, such as refractory status epilepticus, aggressive progression or preventing

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relapses, although variable combination immunotherapy of IVMP, PE and IVIG are utilized,

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there is no or only moderate and short-lived effect for the clinical. Previous studies have demonstrated that multimodality immunotherapy of combination of both first- and second-line therapies are effective

[99, 115-117] .But

it remains not unequivocal which

immunosuppressant may have provided the greatest impact on the outcome since all of the treatments were administered over a short time. Second-line therapy commonly consists

of

Mycophenolate

Cyclophosphamide [12] .Azathioprine

mofetil, and

Azathioprine, mycophenolate

Rituximab, mofetil,

as

and mild

immunosuppressants, are mainly used to maintain remission and reduce dependence on steroids, with varying results. Saidha described two patients with GAD65-epilepsy who

Journal Pre-proof had a partial response to IVIG and prednisolone and successfully improved symptoms with mycophenolate mofetil[116] . In one case report, the patient's condition remained unchanged after 5 years of azathioprine use[118] .In another case report, azathioprine was effective but had a lower clinical response, and was suspended after about 40 days due to severe anemia [114] . Rituximab and cyclophosphamide can be used as first aid therapy in the rapid progression of symptoms, and in some cases, remarkable and lasting

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improvement or remission were observed[80, 115, 117, 119] . But compared to rituximab, it is not

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welcome given the adverse effects of cyclophosphamide. Besides, Triplett reported a

fulminant

encephalopathy

and

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case of GAD65-epilepsy presenting with epilepsia partialis continua progressing to a responding

to

rituximab

combined

with

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cyclophosphamide [87]. Other immunosuppressive agents such as Natalizumab and

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Basiliximab have also been tried without long-term improvement[11, 57] .

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6.2Titer of anti-GAD65 value in guiding immunomodulatory treatment

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anti-GAD65 Abs levels/titers showed poor correlation with the clinical presentation of GAD65-epilepsy, and the value of the change of anti-GAD65 Abs titer in guiding long-term treatment is still unclear. In some cases, serum antibodies remained high titer during and after adequate treatment; however, patients became rapidly asymptomatic without the need for long-term immunomodulatory therapy

[120] .In

some cases, sustained high levels

of anti-GAD65 Abs titers are often associated with clinical adverse effect, and reduced antibody titers are often observed in patients who clinically respond to treatment, but antibodies titers rarely disappear, and often elevated again with clinical symptom recurrence during follow-up.[64,

121] .

Thus, Di Giacomo et al. propose that clinical

Journal Pre-proof responses and "relative" trends in antibodies titers overtime should be used instead of "absolute" values to guide treatment decisions[120] . Furthermore, Gagnon et al. [8] reviewed a total of 58 published cases and did not find a significant difference in outcome between cases whether or not positive anti-GAD65 Abs were reported in CSF. We advocate that the value of anti-GAD65 Abs titer needs to be interpreted in the overall clinical context of the patient under evaluation, patients with positive anti-GAD65 Abs in CSF or high serum

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titers have a higher risk of clinical adverse effect or recurrence. So long-term follow-up

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examinations are necessary and increase the effectiveness of treatment by closely

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monitoring the correlation between changes in clinical symptoms and antibody titers. 6.3Antiepileptic Drugs

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Even though seizures in GAD65-epilepsy are resistant to antiepileptic drugs (AEDs),

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some patients may respond adequately to treatment with only AEDs from the beginning of

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the inflammatory disease or isolated seizures, and some patients can benefit from AEDs

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after Immunotherapy failure[11, 110] . For this reason, AEDs should be used throughout the GAD65-epilepsy. In the Cabezudo-Garcia, et al. Systematic review, 8% of patients with GAD65-epilepsy responded to AEDs and usually required a combination of various AEDs[122] . There is no randomized clinical trial data to support one AED over another ; theoretically, they should be more responsive to AEDs that enhance GABA function . 6.4Epilepsy Surgery The surgical outcomes of GAD65-epilepsy seem to be worse than with other etiologies of refractory epilepsy. In a retrospective study,8 patients with GAD65-associated TLE underwent anterior temporal lobectomy (ATL); 5 patients improve in seizure frequency but

Journal Pre-proof not seizure freedom[123] .In another study, selective amygdalohippocampectomy (SAH) resection for this disease reduced seizures in 2 of 3 patients[11] , but it should be noted that SAH combined with immunotherapy may lead to worse performance of graphics and language memory compared with immunotherapy alone [124] . But, as mentioned above, In the third stage of GAD65-epilepsy, refractory epilepsy appears to be related to underlying "structural" etiology rather than ongoing inflammatory processes. In these conditions,

oo

f

especially when FDG-PET scanning revealed no hypermetabolic areas or serological and

pr

CSF markers have returned to normal, epilepsy surgery can still be considered as a useful

e-

treatment option to reduce seizure frequency and improved quality of life. Additionally, cases successfully treated with vagal stimulation (VNS) or Deep brain stimulation (DBS)

Pr

have been reported. [107, 109] .

anti-GAD65

Abs

levels

have

been

identified

in

many

patients

with

rn

High

al

7.Conclusions

Jo u

acute/subacute-onset, chronic, or cryptogenic epilepsy. Anti-GAD65 Abs are directed against the rate-limiting enzyme for the synthesis of GABA. Although anti-GAD65 Abs have been demonstrated to inhibit GAD65 enzyme activity and reduce amounts of available GABA content in some experiments, the pathogenic role of anti-GAD65 Abs remains unclear due to the intracellular location of the antigen and the controversial evidence of Abs internalization into neurons. Patients with GAD65-epilepsy frequently coexist multiple autoimmune disorders or auto-antibodies and may evolve into a more widespread CNS inflammatory disorder at some point in their illness. This condition often associated with not paraneoplastic, but if co-existing neuronal cell-surface antibodies are

Journal Pre-proof present, the risk of an underlying malignancy will greatly increase. Although patients with GAD65-epilepsy received multiple therapeutic interventions in varying combinations, the efficacy and prognosis were poor. Early identification of anti-GAD65 Abs and aggressive immunosuppressive treatment has the potential to prevent the development of permanent brain tissue damage, but it is usually only a temporary therapeutic success, so more aggressive and long-term immunosuppressive therapies are needed to prevent disease

oo

f

progression and relapse. Improvement of clinical symptoms may parallel a "relative"

pr

reduction in antibody titers, and patients with positive anti-GAD65 Abs in CSF or

e-

extremely high serum titers have a higher risk of clinical adverse effect or recurrence, so monitoring antibodies titers may be used to guide immunotherapy. In the future,

Pr

prospective studies recruiting patients with GAD65-epilepsy are needed to elucidate

al

better the spectrum of epilepsy-related to anti-GAD65 Abs, and further studies on

Jo u

Contributors

rn

underlying pathophysiology may influence therapeutic management strategies.

All authors planned the manuscript, did the literature search, contributed to the f igures, and wrote, edited, and approved the manuscript. F unding This work was supported by the Jilin Province Science and technology development plan project in China (Grant No.20180311075yy) Disclosure of conflicts of interest None of the authors has any conflict of interest to disclose.

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oo

systematic review. Seizure, 2018. 59: p. 72-76.

f

122.

N.,

et

Pre-

al.,

hippocampus-associated

memory

and

long-term

impairment

Pr

Hans en,

postoperative in

epilepsy

courses patients

of with

al

antibody-associated limbic encephalitis and selective amygdalohippocampectomy.

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Epilepsy Behav, 2018. 79: p. 93-99.

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124.

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associated with neuronal antibodies. Epilepsy Res, 2017. 129: p. 101-105.

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comprise

three

domains:

N-terminal,

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GAD67,

e-

Fig. 1. A) Each GAD monomeric unit ， GAD65 and

PLP-binding, and C-terminal domain. 74% identity for the

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middle and C-terminal domains, 25% identity for the

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N-terminal domain. B ） Dimeric structure ofGAD65

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showing the structural locations of the three domains, colored as in Fig. 1(A) Note that ctc1 includes prototypically mAb b78 and some residues from the N-terminal domain, ctc2 includes prototypically mAb 96.11 and some residues from the PLP-domain. (Figure.1. from

Stiff-person

syndrome

(SPS) and

anti-GAD-related CNS degenerations: Protean additions to the autoimmune central neuropathies. Journal of Autoimmunity. 37 (2011) 79-87. copyright 2011; Elsevier. This material is reproduced with permission from Elsevier)

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e-

pr

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f

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A

B

C

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f

D

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Fig. 2. Brain MRI of GAD65-epilepsy. Patient 1. A 61-year-old man experienced left lower

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limb paroxysmal convulsion and numbness, associated with brief heart palpitations, tongue

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stiffness, and sweats several times per day, for two months. Brain MRI: FLAIR sequen ce demonstrating bilateral medial temporal hyperintensities on axial (A) and sagittal (B) sections. Patient 2. A 23-year-old woman presented with left face and tongue twitching,

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therapies

behavioral changes for 20 days, and progressed to confusion and bilateral tonic-clonic seizures 3 days. FLAIR sequence(C) and DWI image (D) demonstrating right temporoparietal lobe slightly hyperintensities on axial.

Table 1. Review of recommending dose, mechanism of action, adverse effects of various immune therapies: First-line and Second-line Recommended dose Fi rst-line therapies

Mechanism of action

Adverse effects

Journal Pre-proof

dose:

IV.

Modulates

methylprednisolone

and

(MP)

production,

pulses.

1000

on

3–5

mg/day

consecutive days ↓ 1000

mg/day

on

consecutive

chemokine cytokine adhesion

molecule

expression

Insomnia,

psychiatric

symptoms, hyperglycemia, hypertension

obesity,

electrolyte

imbalances,

and reduces migration

hypertension, peptic ulcer,

of leukocytes to the

Cushing

target tissue

cataracts,

syndrome,

3

f

Initial

oo

Corticosteroids

days

infection

osteoporosis,

once

weekly for 5 weeks or mg/day

consecutive

days by

3 ，

once

al

followed

on

necrosis

Pr

1000

pr

by

avascular

e-

followed

and

rn

weekly for 5 weeks,

Jo u

then every 2 weeks for 6 weeks.

Oral.

60–80

prednisone duration

↓

of

mg daily,

tapering

dose IVIG

Initial

dose:

0.4/kg

body-weight/day on 3–5

Modulates complement

Anaphylactic

activation,

(IgA-deficient patients) acute

suppress

reactions

Journal Pre-proof

consecutive days

idiotypic

↓

antibodies,

renal failure, thromboembolic

saturate Fc receptors

events, hemolytic anemia,

on macrophages, and

aseptic meningitis，headache

0.4/kg body-weight/day on 3 consecutive days suppress followed

by

cytokines,

once chemokines,

and

weekly for 5 weeks or metalloproteinases

oo

f

0.4/kg body-weight/day on 3 consecutive days once

weekly for 5 weeks,

Pr

then every 2 weeks for 6 weeks.

An extracorporeal blood

Hypotension and electrolyte

day spread over 5–7

purification technique，

imbalance,

cycles,10–14 days

removes

hemorrhage,

al

1 exchange every other

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rn

Plasmapheresis

pr

by

e-

followed

large

molecular-weight molecules,

such

as

complex,

complements and filters out plasma membrane proteins

thrombosis,

and pneumothorax

immunoglobulins, immune

infection,

Journal Pre-proof

Se cond-line therapies 500–800

mg/m2/

An

alkylating

interferes with

symptoms,

which

Oral: 1–2 mg/kg/day

DNA synthesis, causing

failure,

neutropenia,

cell death by DNA–RNA

infections,

hemorrhagic

cross-linking

cystitis

and

of

alopecia, mucositis, ovarian

protein

IV: monthly single day

A humanized anti–α4

Progressive

infusions of 300 mg

e-

pr

synthesis

leukoencephalopathy (PML),

integrin

monoclonal

gastrointestinal

entry of T and B cells

allergic

al

Pr

antibody that prevents

rn

into the CNS.

multifocal

symptoms,

reaction,

liver

problems, headache, tired feeling, cold symptoms, joint

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Gastrointestinal

month for 3–6 months

inhibition

Natalizumab

agent

f

de

IV:

oo

Cyclophosphami

pain,

vaginal

itching

or

discharge

IV: 1000 mg followed

A

humanized

Allergic reaction, infections,

by once weekly for 2

monoclonal IgG against

reactivation of tuberculosis

weeks, or 375 mg/m 2

CD20-positive B cells

infection,

weekly for 4 weeks.

which leads to B cell

infection,

depletion and reduces

neutropenia.

autoantibody

or

hepatitis

B

late-onset

Journal Pre-proof

generation Mycophenolate

Oral: Initially 500 mg

Inhibition

of

mofetil

twice daily, target 1000

monophosphate

hypertension,

mg twice daily.

dehydrogenase

hypercholesterolemia,

de

novo

Gastrointestinal

symptoms,

hepatotoxicity,

peripheral

synthesis of guanosine

edema,

infections,

nucleotides, leading to

myelosuppression,

selective inhibition of

lymphoma,

pr

oo

f

mediated

inosine

other

malignancies

e-

lymphocyte

and

Oral:

Initially

1.5

rn

mg/kg/ day

Converted to cytotoxic

Gastrointestinal

symptoms,

6-thioguanine

hypersensitivity

reactions,

al

mg/kg/day, target 2.5–3

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Azathioprine

Pr

proliferation.

nucleotides

which

intercalates

into

lymphopenia, hepatotoxicity,

and

opportunistic infection.

replicating

DNA

can block the de novo pathway

of

purine

synthesis lymphocyte hair

loss,

inducing apoptosis cytopenia,

hepatotoxicity, lymphoma,

and

alopecia,

fatigue,

Journal Pre-proof

infections Basiliximab

IV :20 mg/month

An

interleukin-2

Allergic

receptor Ab that inhibits

lymphopenia,

T

infection

cell-mediated

reaction, opportunistic

immune reactions.

oo

Anti-GAD65 Abs were found in patients with seizures in the context of encephalitis,

pr



f

Highlights:

e-

and patients with chronic epileptic syndromes.

The pathogenic role of anti-GAD65 Abs remains unclear



Immune-mediated epilepsy with GAD65 antibodies frequently coexist multiple

Pr



al

autoimmune disorders or auto-antibodies and may evolve into a more widespread

The evidence of high serum titers and intrathecal synthesis play a fundamental role in

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

rn

CNS inflammatory disorder.

diagnosis but poorly correlate with disease severity or response to therapies. 

Early immunosuppressive therapy can achieve temporary success, more aggressive and long-term immunosuppressive therapies may be needed to prevent disease progression and relapse.

Table 1. Review of recommending dose, mechanism of action, adverse effects of various immune therapies: First -line and Second-line therapies