Characterisation of TRIM46 autoantibody-associated paraneoplastic neurological syndrome.

OBJECTIVES: To report the expanded neurological presentations and oncological associations of tripartite motif-containing protein 46 (TRIM46)-IgG seropositive patients. METHODS: Archived sera/cerebrospinal fluid (CSF) were evaluated by tissue-based immunofluorescence assay to identify patients with identical axon initial segment (AIS)-specific staining pattern. Phage immunoprecipitation sequencing (PhIP-Seq) was used to identify the putative autoantigen. RESULTS: IgG in serum (17) and/or CSF (16) from 25 patients yielded unique AIS-specific staining on murine central nervous system (CNS) tissue. An autoantibody specific for TRIM46 was identified by PhIP-Seq, and autoantigen specificity was confirmed by transfected COS7 cell-based assay. Clinical information was available for 22 TRIM46-IgG seropositive patients. Fifteen were female (68%). Median age was 67 years (range 25-87). Fifteen (68%) patients presented with subacute cerebellar syndrome (six isolated; nine with CNS accompaniments: encephalopathy (three), brainstem signs (two), myelopathy (two), parkinsonism (one)). Other phenotypes included limbic encephalitis (three), encephalopathy with/without seizures (two), myelopathy (two). Eighteen (82%) had cancer: neuroendocrine carcinomas (9; pancreatic (3), small-cell lung (4), oesophagus (1), endometrium (1)), adenocarcinomas (6; lung (2), ovarian (2), endometrial (1), breast (1)), sarcoma (2) and gastrointestinal tumour (1). Neurological symptoms in three followed immune checkpoint inhibitor (ICI) administration. CONCLUSIONS: This study supports TRIM46-IgG being a biomarker of paraneoplastic CNS disorders and expands the neurological phenotypes, oncological and ICI-related adverse event associations.

Neurochondrin neurological autoimmunity.

OBJECTIVES: To describe the neurologic spectrum and treatment outcomes for neurochondrin-IgG positive cases identified serologically in the Mayo Clinic Neuroimmunology Laboratory. METHODS: Archived serum and CSF specimens previously scored positive for IgGs that stained mouse hippocampal tissue in a nonuniform synaptic pattern by immunofluorescence assay (89 among 616,025 screened, 1993-2019) were reevaluated. Antibody characterization experiments revealed specificity for neurochondrin, confirmed by recombinant protein assays. RESULTS: IgG in serum (9) or CSF (4) from 8 patients yielded identical neuron-restricted CNS patterns, most pronounced in hippocampus (stratum lucidum in particular), cerebellum (Purkinje cells and molecular layer), and amygdala. All were neurochondrin-IgG positive. Five were women; median symptom onset age was 43 years (range, 30-69). Of 7 with clinical data, 6 presented with rapidly progressive cerebellar ataxia, brainstem signs, or both; 1 had isolated unexplained psychosis 1 year prior. Five of 6 had cerebellar signs, 4 with additional brainstem symptoms or signs (eye movement abnormalities, 3; dysphagia, 2; nausea and vomiting, 1). One patient with brainstem signs (vocal cord paralysis and VII nerve palsy) had accompanying myelopathy (longitudinally extensive abnormality on MRI; aquaporin-4-IgG and myelin oligodendrocyte glycoprotein-IgG negative). The 7th patient had small fiber neuropathy only. Just 1 of 7 had contemporaneous cancer (uterine). Six patients with ataxia or brainstem signs received immunotherapy, but just 1 remained ambulatory. At last follow-up, 5 had MRI evidence of severe cerebellar atrophy. CONCLUSION: In our series, neurochondrin autoimmunity was usually accompanied by a nonparaneoplastic rapidly progressive rhombencephalitis with poor neurologic outcomes. Other phenotypes and occasional paraneoplastic causes may occur.

Autoimmune gait disturbance accompanying adaptor protein-3B2-IgG.

OBJECTIVE: To describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein-3 (AP3). METHODS: Archived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin-immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively. RESULTS: Serum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24-58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3-94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA). CONCLUSION: AP3B2 (previously named ß-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable.

Kelch-like Protein 11 Antibodies in Seminoma-Associated Paraneoplastic Encephalitis.

A 37-year-old man with a history of seminoma presented with vertigo, ataxia, and diplopia. An autoantibody specific for kelch-like protein 11 (KLHL11) was identified with the use of programmable phage display. Immunoassays were used to identify KLHL11 IgG in 12 other men with similar neurologic features and testicular disease. Immunostaining of the patient's IgG on mouse brain tissue showed sparse but distinctive points of staining in multiple brain regions, with enrichment in perivascular and perimeningeal tissues. The onset of the neurologic syndrome preceded the diagnosis of seminoma in 9 of the 13 patients. An age-adjusted estimate of the prevalence of autoimmune KLHL11 encephalitis in Olmsted County, Minnesota, was 2.79 cases per 100,000 men. (Funded by the Rochester Epidemiology Project and others.).

Paraneoplastic neuronal intermediate filament autoimmunity.

OBJECTIVE: To describe paraneoplastic neuronal intermediate filament (NIF) autoimmunity. METHODS: Archived patient and control serum and CSF specimens were evaluated by tissue-based indirect immunofluorescence assay (IFA). Autoantigens were identified by Western blot and mass spectrometry. NIF specificity was confirmed by dual tissue section staining and 5 recombinant NIF-specific HEK293 cell-based assays (CBAs, for α-internexin, neurofilament light [NfL], neurofilament medium, or neurofilament heavy chain, and peripherin). NIF-immunoglobulin Gs (IgGs) were correlated with neurologic syndromes and cancers. RESULTS: Among 65 patients, NIF-IgG-positive by IFA and CBAs, 33 were female (51%). Median symptom onset age was 62 years (range 18-88). Patients fell into 2 groups, defined by the presence of NfL-IgG (21 patients, who mostly had ≥4 NIF-IgGs detected) or its absence (44 patients, who mostly had ≤2 NIF-IgGs detected). Among NfL-IgG-positive patients, 19/21 had ≥1 subacute onset CNS disorders: cerebellar ataxia (11), encephalopathy (11), or myelopathy (2). Cancers were detected in 16 of 21 patients (77%): carcinomas of neuroendocrine lineage (10) being most common (small cell [5], Merkel cell [3], other neuroendocrine [2]). Two of 257 controls (0.8%, both with small cell carcinoma) were positive by both IFA and CBA. Five of 7 patients with immunotherapy data improved. By comparison, the 44 NfL-IgG-negative patients had findings of unclear significance: diverse nervous system disorders (p = 0.006), as well as limited (p = 0.003) and more diverse (p < 0.0001) cancer accompaniments. CONCLUSIONS: NIF-IgG detection by IFA, with confirmatory CBA testing that yields a profile including NfL-IgG, defines a paraneoplastic CNS disorder (usually ataxia or encephalopathy) accompanying neuroendocrine lineage neoplasia.

Autoimmune septin-5 cerebellar ataxia.

OBJECTIVE: To report a form of autoimmune cerebellar ataxia in which antibodies target septin-5, a guanosine triphosphate (GTP)-binding neural protein involved in neurotransmitter exocytosis. METHODS: Archived sera and CSF specimens with unclassified synaptic antibodies were re-evaluated by tissue-based indirect immunofluorescence assay. Autoantigens were identified by Western blot and mass spectrometry. Recombinant protein assays (Western blot, cell based, and protein screening array) confirmed antigen specificity. RESULTS: Serum and CSF from 6 patients produced identical synaptic immunoglobulin G (IgG) staining patterns of synaptic regions (neuropil) of the mouse cerebrum and cerebellum. The molecular layer of the cerebellum and the thalamus demonstrated stronger immunoreactivity than the midbrain, hippocampus, cortex, and basal ganglia. The antigen revealed by mass spectrometry analysis of immunoprecipitated cerebellar proteins and confirmed by recombinant protein assays was septin-5. All 4 patients with records available had subacute onset of cerebellar ataxia with prominent eye movement symptoms (oscillopsia or vertigo). None had cancer detected. Improvements occurred after immunotherapies (2) or spontaneously (1). One patient died. CONCLUSION: Septin-5 IgG represents a biomarker for a potentially fatal but treatable autoimmune ataxia.

Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

Neuromyelitis optica (NMO) is an inflammatory disorder mediated by antibodies to aquaporin-4 (AQP4) with prominent blood-brain barrier (BBB) breakdown in the acute phase of the disease. Anti-AQP4 antibodies are produced mainly in the periphery, yet they target the astrocyte perivascular end feet behind the BBB. We reasoned that an endothelial cell-targeted autoantibody might promote BBB transit of AQP4 antibodies and facilitate NMO attacks. Using monoclonal recombinant antibodies (rAbs) from patients with NMO, we identified two that strongly bound to the brain microvascular endothelial cells (BMECs). Exposure of BMECs to these rAbs resulted in nuclear translocation of nuclear factor κB p65, decreased claudin-5 protein expression, and enhanced transit of macromolecules. Unbiased membrane proteomics identified glucose-regulated protein 78 (GRP78) as the rAb target. Using immobilized GRP78 to deplete GRP78 antibodies from pooled total immunoglobulin G (IgG) of 50 NMO patients (NMO-IgG) reduced the biological effect of NMO-IgG on BMECs. GRP78 was expressed on the surface of murine BMECs in vivo, and repeated administration of a GRP78-specific rAb caused extravasation of serum albumin, IgG, and fibrinogen into mouse brains. Our results identify GRP78 antibodies as a potential component of NMO pathogenesis and GRP78 as a candidate target for promoting central nervous system transit of therapeutic antibodies.

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

Microtubule-associated protein 1B: Novel paraneoplastic biomarker.

OBJECTIVE: To report the identification of microtubule-associated protein (MAP) 1B as the antigen of the previously described Purkinje cell cytoplasmic antibody type 2 (PCA-2) antibody, its frequency, and clinical, oncological, and serological associations. METHODS: Archival serum or cerebrospinal fluid (CSF) specimens were available from 96 of 118 consecutive PCA-2-IgG-seropositive patients identified during 1993-2016. The autoantigen, defined in mouse brain lysate by Western blot and mass spectrometry, was confirmed by dual immunohistochemical staining using commercial antibodies. The major antigenic region was defined by Western blot using recombinant protein fragments. RESULTS: IgG in 95 of 96 patients' serum or CSF (but in none of 98 healthy or disease control subjects' serum specimens) bound to recombinant MAP1B. A minority (17.5%) of patients' IgG also bound to MAP1A. PCA-2 was often accompanied by additional neural autoantibody markers of small-cell carcinoma, including collapsin response-mediated protein 5 (CRMP5) IgG (26%) or antineuronal nuclear antibody type 1 (ANNA-1) IgG (also known as anti-Hu; 13%). Neurological manifestations in 95 patients were (in decreasing frequency): peripheral neuropathy, 53%; cerebellar ataxia, dysmetria, or dysarthria, 38%; and encephalopathy, 27%. Cancer (majority small-cell lung carcinoma [SCLC]) was detected in 66 of 84 evaluated patients (79%). The MAP1B (PCA-2) autoantibody detection rate, among approximately 70,000 patients undergoing service neural autoantibody evaluation in 2015, was 0.024%, equaling amphiphysin IgG (0.026%) and more common than ANNA-2 (also known as anti-Ri; 0.016%) and PCA-Tr (also known as delta/notch-like epidermal growth factor-related receptor [DNER]; 0.006%). INTERPRETATION: MAP1B, the PCA-2 autoantigen, represents a novel target in paraneoplastic neurological disorders and has high predictive value for SCLC. Its relatively high prevalence, compared with other recognized paraneoplastic neural autoantibodies, justifies its testing in comprehensive paraneoplastic neural autoantibody evaluation. Ann Neurol 2017;81:266-277.

Effects of neuromyelitis optica-IgG at the blood-brain barrier in vitro.

OBJECTIVE: To address the hypothesis that physiologic interactions between astrocytes and endothelial cells (EC) at the blood-brain barrier (BBB) are afflicted by pathogenic inflammatory signaling when astrocytes are exposed to aquaporin-4 (AQP4) antibodies present in the immunoglobulin G (IgG) fraction of serum from patients with neuromyelitis optica (NMO), referred to as NMO-IgG. METHODS: We established static and flow-based in vitro BBB models incorporating co-cultures of conditionally immortalized human brain microvascular endothelial cells and human astrocyte cell lines with or without AQP4 expression. RESULTS: In astrocyte-EC co-cultures, exposure of astrocytes to NMO-IgG decreased barrier function, induced CCL2 and CXCL8 expression by EC, and promoted leukocyte migration under flow, contingent on astrocyte expression of AQP4. NMO-IgG selectively induced interleukin (IL)-6 production by AQP4-positive astrocytes. When EC were exposed to IL-6, we observed decreased barrier function, increased CCL2 and CXCL8 expression, and enhanced leukocyte transmigration under flow. These effects were reversed after application of IL-6 neutralizing antibody. CONCLUSIONS: Our results indicate that NMO-IgG induces IL-6 production by AQP4-positive astrocytes and that IL-6 signaling to EC decreases barrier function, increases chemokine production, and enhances leukocyte transmigration under flow.

Autoimmune Glial Fibrillary Acidic Protein Astrocytopathy: A Novel Meningoencephalomyelitis.

IMPORTANCE: A novel astrocytic autoantibody has been identified as a biomarker of a relapsing autoimmune meningoencephalomyelitis that is immunotherapy responsive. Seropositivity distinguishes autoimmune glial fibrillary acidic protein (GFAP) meningoencephalomyelitis from disorders commonly considered in the differential diagnosis. OBJECTIVE: To describe a novel IgG autoantibody found in serum or cerebrospinal fluid that is specific for a cytosolic intermediate filament protein of astrocytes. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of the medical records of seropositive patients identified in the Mayo Clinic Neuroimmunology Laboratory from October 15, 1998, to April 1, 2016, in blinded comprehensive serologic evaluation for autoantibody profiles to aid the diagnosis of neurologic autoimmunity (and predict cancer likelihood). MAIN OUTCOMES AND MEASURES: Frequency and definition of novel autoantibody, the autoantigen's immunochemical identification, clinical and magnetic resonance imaging correlations of the autoantibody, and immunotherapy responsiveness. RESULTS: Of 103 patients whose medical records were available for review, the 16 initial patients identified as seropositive were the subject of this study. Median age at neurologic symptom onset was 42 years (range, 21-73 years); there was no sex predominance. The novel neural autoantibody, which we discovered to be GFAP-specific, is disease spectrum restricted but not rare (frequency equivalent to Purkinje cell antibody type 1 [anti-Yo]). Its filamentous pial, subventricular, and perivascular immunostaining pattern on mouse tissue resembles the characteristic magnetic resonance imaging findings of linear perivascular enhancement in patients. Prominent clinical manifestations are headache, subacute encephalopathy, optic papillitis, inflammatory myelitis, postural tremor, and cerebellar ataxia. Cerebrospinal fluid was inflammatory in 13 of 14 patients (93%) with data available. Neoplasia was diagnosed within 3 years of neurologic onset in 6 of 16 patients (38%): prostate and gastroesophageal adenocarcinomas, myeloma, melanoma, colonic carcinoid, parotid pleomorphic adenoma, and teratoma. Neurologic improvement followed treatment with high-dose corticosteroids, with a tendency of patients to relapse without long-term immunosuppression. CONCLUSIONS AND RELEVANCE: Glial fibrillary acidic protein-specific IgG identifies a distinctive, corticosteroid-responsive, sometimes paraneoplastic autoimmune meningoencephalomyelitis. It has a lethal canine equivalent: necrotizing meningoencephalitis. Expression of GFAP has been reported in some of the tumor types identified in paraneoplastic cases. Glial fibrillary acidic protein peptide-specific cytotoxic CD8+ T cells are implicated as effectors in a transgenic mouse model of autoimmune GFAP meningoencephalitis.

Signal recognition particle immunoglobulin g detected incidentally associates with autoimmune myopathy.

INTRODUCTION: Paraneoplastic autoantibody screening of 150,000 patient sera by tissue-based immunofluorescence incidentally revealed 170 with unsuspected signal recognition particle (SRP) immunoglobulin G (IgG), which is a recognized biomarker of autoimmune myopathy. Of the 77 patients with available information, 54 had myopathy. We describe the clinical/laboratory associations. METHODS: Distinctive cytoplasm-binding IgG (mouse tissue substrate) prompted western blot, enzyme-linked immunoassay, and immunoprecipitation analyses. Available histories were reviewed. RESULTS: The immunostaining pattern resembled rough endoplasmic reticulum, and mimicked Purkinje-cell cytoplasmic antibody type 1 IgG/anti-Yo. Immunoblotting revealed ribonucleoprotein reactivity. Recombinant antigens confirmed the following: SRP54 IgG specificity alone (17); SRP72 IgG specificity alone (3); both (32); or neither (2). Coexisting neural autoantibodies were identified in 28% (low titer). Electromyography revealed myopathy with fibrillation potentials; 78% of biopsies had active necrotizing myopathy with minimal inflammation, and 17% had inflammatory myopathy. Immunotherapy responsiveness was typically slow and incomplete, and relapses were frequent on withdrawal. Histologically confirmed cancers (17%) were primarily breast and hematologic, with some others. CONCLUSIONS: Autoimmune necrotizing SRP myopathy, both idiopathic and paraneoplastic, is underdiagnosed in neurological practice. Serological screening aids early diagnosis. Cancer surveillance and appropriate immunosuppressant therapy may improve outcome. Muscle Nerve 53: 925-932, 2016.

The neurologic significance of celiac disease biomarkers.

OBJECTIVE: To report neurologic phenotypes and their etiologies determined among 68 patients with either (1) celiac disease (CD) or (2) no CD, but gliadin antibody positivity (2002-2012). METHODS: Neurologic patients included both those with the CD-prerequisite major histocompatibility complex class II human leukocyte antigen (HLA)-DQ2/DQ8 haplotype, and those without. The 3 groups were as follows: group 1 (n = 44), CD or transglutaminase (Tg)-2/deamidated gliadin immunoglobulin (Ig)A/IgG detected; group 2 (n = 15), HLA-DQ2/DQ8 noncarriers, and gliadin IgA/IgG detected; and group 3 (n = 9), HLA-DQ2/DQ8 carriers, and gliadin IgA/IgG detected. Neurologic patients and 21 nonneurologic CD patients were evaluated for neural and Tg6 antibodies. RESULTS: In group 1, 42 of 44 patients had CD. Neurologic phenotypes (cerebellar ataxia, 13; neuropathy, 11; dementia, 8; myeloneuropathy, 5; other, 7) and causes (autoimmune, 9; deficiencies of vitamin E, folate, or copper, 6; genetic, 6; toxic or metabolic, 4; unknown, 19) were diverse. In groups 2 and 3, 21 of 24 patients had cerebellar ataxia; none had CD. Causes of neurologic disorders in groups 2 and 3 were diverse (autoimmune, 4; degenerative, 4; toxic, 3; nutritional deficiency, 1; other, 2; unknown, 10). One or more neural-reactive autoantibodies were detected in 10 of 68 patients, all with autoimmune neurologic diagnoses (glutamic acid decarboxylase 65 IgG, 4; voltage-gated potassium channel complex IgG, 3; others, 5). Tg6-IgA/IgG was detected in 7 of 68 patients (cerebellar ataxia, 3; myelopathy, 2; ataxia and parkinsonism, 1; neuropathy, 1); the 2 patients with myelopathy had neurologic disorders explained by malabsorption of copper, vitamin E, and folate rather than by neurologic autoimmunity. CONCLUSIONS: Our data support causes alternative to gluten exposure for neurologic dysfunction among most gliadin antibody-positive patients without CD. Nutritional deficiency and coexisting autoimmunity may cause neurologic dysfunction in CD.

Investigation of the KIR4.1 potassium channel as a putative antigen in patients with multiple sclerosis: a comparative study.

BACKGROUND: Antibodies have been implicated in the pathogenicity of multiple sclerosis by findings of immunoglobulins in patients' CSF and often IgG and complement in lesions, and by a 2012 report that nearly half of patients' serum samples contain IgG specific for a glial potassium-channel, KIR4.1. We aimed to establish the frequency of KIR4.1-binding IgG in serum and CSF of patients with multiple sclerosis, and whether KIR4.1 immunoreactivity is retained or lost in demyelinating lesions. METHODS: Using ELISA with a KIR4.1 peptide, we tested archival serum from 229 population-based and 57 clinic-based patients with multiple sclerosis, 99 healthy controls, and 109 disease controls, and CSF from 25 patients with multiple sclerosis and 22 disease controls. We tested all CSF and serum samples from 50 of the clinic-based patients with multiple sclerosis on cells expressing functional KIR4.1, using cell-based immunofluorescence and immunoprecipitation (solubilised recombinant human KIR4.1). We assessed KIR4.1 immunoreactivity in archival brain samples from 15 patients with histopathologically confirmed multiple sclerosis (22 plaques [eight early active, eight inactive, and six remyelinated], 13 periplaque regions and eight normal-appearing white-matter and grey-matter regions) and from three controls with non-neurological diseases. FINDINGS: Three of 286 serum samples from patients with multiple sclerosis and two of 208 serum samples from controls showed KIR4.1 reactivity on ELISA; none of the CSF samples from patients or controls showed KIR4.1 reactivity. IgG in none of the 50 serum samples from clinic-based patients immunoprecipitated KIR4.1, but a commercial KIR4.1-specific control IgG did. By immunofluorescence, one of 50 serum samples from patients with multiple sclerosis yielded faint plasmalemmal staining on both KIR4.1-expressing and non-expressing cells; 16 bound faintly to intracellular components. In all cases, IgG binding was quenched by absorption with liver powder or lysates from non-transfected cells. Binding by the KIR4.1-specific control IgG was quenched only by lysates containing KIR4.1. IgG in none of the 25 CSF samples from patients with multiple sclerosis bound to KIR4.1-transfected cells. Glial KIR4.1 immunoreactivity was increased relative to expression in healthy control brain in all active demyelinating lesions, remyelinated lesions, and periplaque white matter regions. INTERPRETATION: We did not detect KIR4.1-specific IgG in serum or CSF from patients with multiple sclerosis or KIR4.1 loss from glia in multiple sclerosis lesions. Serological testing for KIR4.1-specific IgG is unlikely to aid diagnosis of multiple sclerosis. The target antigen of multiple sclerosis remains elusive. FUNDING: The National Institutes of Health, the National Multiple Sclerosis Society, and the Mayo Clinic Robert and Arlene Kogod Center on Aging.

Potassium channel complex autoimmunity induced by inhaled brain tissue aerosol.

OBJECTIVE: To test the hypothesis that autoimmunity induced by inhalation of aerosolized brain tissue caused outbreaks of sensory-predominant polyradiculoneuropathy among swine abattoir employees in the Midwestern United States. METHODS: Mice were exposed intranasally, 5 days per week, to liquefied brain tissue. Serum from exposed mice, patients, and unaffected abattoir employees were analyzed for clinically pertinent neural autoantibodies. RESULTS: Patients, coworkers, and mice exposed to liquefied brain tissue had an autoantibody profile dominated by neural cation channel immunoglobulin Gs (IgGs). The most compelling link between patients and exposed mice was magnetic resonance imaging (MRI) evidence of grossly swollen spinal nerve roots. Autoantibody responses in patients and mice were dose-dependent and declined after antigen exposure ceased. Autoantibodies detected most frequently, and at high levels, bound to detergent-solubilized macromolecular complexes containing neuronal voltage-gated potassium channels ligated with a high affinity Kv1 channel antagonist, 125I-α-dendrotoxin. Exposed mice exhibited a behavioral phenotype consistent with potassium channel dysfunction recognized in drosophila with mutant ("shaker") channels: reduced sensitivity to isoflurane-induced anesthesia. Pathological and electrophysiological findings in patients supported peripheral nerve hyperexcitability over destructive axonal loss. The pain-predominant symptoms were consistent with sensory nerve hyperexcitability. INTERPRETATION: Our observations establish that inhaled neural antigens readily induce neurological autoimmunity and identify voltage-gated potassium channel complexes as a major immunogen.

Eliminating false-positive results in serum tests for neuromuscular autoimmunity.

In this study we describe the false-positive frequency in radioimmunoprecipitation assays for muscle acetylcholine receptor (AChR) and neuronal voltage-gated potassium channel (VGKC) autoantibodies, attributable to 125I-ligand immunoprecipitation. Sera were evaluated for AChR autoantibody (n = 34,095) and VGKC autoantibody (n = 11,028). We retested sera that yielded apparently positive results with 125I-ligand with and without detergent-solubilized cation-channel protein, indentified clinically validated fals-positive rates of 0.05% and 1.7% for AchR and VGKC autoantibodies, respectively. Specificity assurance in radioimmunoprecipitation assays requires subtraction of values for 125I-ligand binding.

Peripherin-IgG association with neurologic and endocrine autoimmunity.

Peripherin-IgG has been reported a pertinent autoantibody in non-obese type 1 diabetic (NOD) mice. However, it has not previously been recognized in any human disease. In blinded evaluation of serum for markers of neurological autoimmunity in a high-volume diagnostic laboratory, we incidentally identified 26 patients (61% female) with an IgG that bound selectively to neural elements in enteric ganglia, sympathetic nerve trunks and discrete nerve tracts in mid-brain and hind-brain. The target antigen was identified as peripherin, a 55kDa - type III intermediate filament protein. Review of clinical histories revealed that 54% of seropositive patients had dysautonomia (predominantly gastrointestinal dysmotility), 30% had neuropathies with varied sensory symptoms and 35% had clinical or serological evidence of endocrinopathy (type 1 diabetes, thyroiditis or premature ovarian failure). Collectively, 73% had autonomic dysfunction or endocrinopathy. None of 173 healthy subjects was seropositive. Subsequent western blot evaluation of archival sera from patients with small fiber/autonomic neuropathies (with or without endocrinopathy) revealed a 33% seropositivity rate for peripherin-IgG. Our further demonstration that peripherin-immunoreactive autonomic fibers in pancreas, thyroid and ovary are juxtaposed to endocrine epithelium, complement our clinical observations in suggesting that neuronal elements may be a pertinent initial target for immune attack in multiple forms of endocrine autoimmunity (intermolecular epitope spreading). It remains to be determined whether or not peripherin-IgG is predictive for development of small fiber neuropathy (autonomic or somatic).

Diagnosis of neuromyelitis spectrum disorders: comparative sensitivities and specificities of immunohistochemical and immunoprecipitation assays.

OBJECTIVE: To compare the sensitivity and specificity of immunofluorescence (IF) and immunoprecipitation (IP) assays using green fluorescent protein-tagged aquaporin-4 (AQP4) in 6335 patients for whom serological evaluation was requested on a service basis. DESIGN: Case-control study. SETTING: Mayo Clinic Neuroimmunology Laboratory (Rochester, Minnesota) and Departments of Neurology (Rochester, Minnesota; Scottsdale, Arizona; and Jacksonville, Florida). Patients Group 1, 835 Mayo Clinic patients, 100 with a neuromyelitis optica (NMO) spectrum disorder diagnosis and 735 without NMO spectrum disorder; group 2, 5500 non-Mayo Clinic patients. Main Outcome Measure Sensitivity and specificity of each assay for NMO or NMO spectrum disorder, individually and combined. RESULTS: In group 1, the sensitivity rates for NMO were IF, 58%; IP, 33%; and combined assays, 63%. The sensitivity rates for relapsing longitudinally extensive transverse myelitis were IF, 29%; IP, 6%; and combined assays, 29%. The specificity rates for NMO and relapsing longitudinally extensive transverse myelitis were IF, 99.6%; IP, 99.3%; and combined assays, 99.2%. In group 2, NMO-IgG was detected by IF in 498 of 5500 patients (9.1%) and by IP in 331 patients (6.0%); 76 of the 331 patients seropositive by IP (23%) were negative by IF. Clinical information was available for 124 patients (including 16 of those seropositive by IP only); 123 had a definite NMO spectrum disorder and 1 was at risk for NMO (monophasic optic neuritis). CONCLUSIONS: In this large, clinical practice-based study, NMO-IgG detected by IF or IP was highly specific for NMO spectrum disorders. The IP assay was significantly less sensitive than IF. Combined testing improved sensitivity by 5%.

Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2.

Neuromyelitis optica (NMO)-immunoglobulin G (IgG) is a clinically validated serum biomarker that distinguishes relapsing central nervous system (CNS) inflammatory demyelinating disorders related to NMO from multiple sclerosis. This autoantibody targets astrocytic aquaporin-4 (AQP4) water channels. Clinical, radiological, and immunopathological data suggest that NMO-IgG might be pathogenic. Characteristic CNS lesions exhibit selective depletion of AQP4, with and without associated myelin loss; focal vasculocentric deposits of IgG, IgM, and complement; prominent edema; and inflammation. The effect of NMO-IgG on astrocytes has not been studied. In this study, we demonstrate that exposure to NMO patient serum and active complement compromises the membrane integrity of CNS-derived astrocytes. Without complement, astrocytic membranes remain intact, but AQP4 is endocytosed with concomitant loss of Na(+)-dependent glutamate transport and loss of the excitatory amino acid transporter 2 (EAAT2) . Our data suggest that EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex. Transport-competent EAAT2 protein is up-regulated in differentiating astrocyte progenitors and in nonneural cells expressing AQP4 transgenically. Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data. Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

Glutamic acid decarboxylase autoimmunity with brainstem, extrapyramidal, and spinal cord dysfunction.

OBJECTIVE: To describe novel neurological manifestations associated with glutamic acid decarboxylase (GAD65) autoimmunity. PATIENTS AND METHODS: This retrospective study (1987-2003) describes 62 patients Incidentally found to have a serum autoantibody that bound selectively to synapse-rich central nervous system tissues. The immunostaining pattern was determined to be GAD65-specific by radiolmmunoprecipitation assay. These cases were identified among samples submitted for paraneoplastic autoantibody evaluation using indirect immunofluorescence. In no case had GAD65 or any other islet cell antibody testing been requested. RESULTS: In most cases, the patients' presentations were initially considered neurodegenerative or inflammatory (multiple sclerosis or paraneoplastic). Median age at onset was 50 years, and 77% were women. Of the 44 patients seen at the Mayo Clinic, 23% were African American; in contrast, less than 10% of Mayo Clinic's neurology patients are African American. Median follow-up was 24 months. The radioimmunoprecipitation assay values for GAD65 antibody were extremely high (median, 1429 nmol/L; Interquartile range, 643-3078 nmol/L) and correlated significantly with immunofluorescence titers (median, 3840; interquartile range, 1920-15,360; r = 0.81; P < .001). Neurological manifestations were multifocal in 41 patients and included cerebellar ataxia (63%), brainstem involvement (29%), seizures (27%), stiff-man phenomena (26%), extrapyramidal signs (16%), and myelopathy (8%). One third of the patients had type 1 diabetes mellitus, 53% had thyroid autoantibodies, and 16% had vitiligo. Eleven of 20 patients identified as African American had brainstem involvement. Some patients appeared to benefit from short-term immunosuppression (none received long-term therapy). CONCLUSIONS: The neurological spectrum of GAD65 autoimmunity includes brainstem, extrapyramidal, and spinal cord syndromes. In our experience, African American patients were disproportionately affected. A patient with a presumed neurodegenerative disorder of new onset, with high levels of GAD65 antibody (>20 nmol/L), merits consideration of immunotherapy.