The expanded clinical spectrum of anti-GABABR encephalitis and added value of KCTD16 autoantibodies

In this study we report the clinical features of 32 patients with gamma aminobutyric acid B receptor (GABABR) antibodies, identify additional autoantibodies in patients with anti-GABABR encephalitis that mark the presence of an underlying small cel

N-Methyl-D-Aspartate receptor antibodies in neuroinflammatory diseases

Die neurologische Autoimmunerkrankung N-Methyl-D-Aspartat Rezeptor (NMDAR) Enzephalitis wird durch den Nachweis spezifischer NMDAR-Autoantikörper diagnostiziert. Zell-basierte Testmethoden oder immunhistochemische Färbungen von Rattenhirngewebe sind zuverlässige Testmethoden, allerdings sind diese Antikörpertests kontroversiell diskutiert. In dieser Studie wurde ein Immunfluoreszenz-Test basierend auf lebenden HEK293A-Zellen, die funktionelle NMDAR exprimieren (CBA), mit einer Durchflusszytometrie (FACS)-basierten Testmethode verglichen. Aufgrund einer möglichen Assoziation von NMDAR Antikörpern mit demyelinisierenden Erkrankungen wurde außerdem die Häufigkeit dieser Antikörper in einer PatientInnenkohorte mit diesen Erkrankungen untersucht. Beide Testmethoden wurden mit einer Testgruppe, bestehend aus Serumproben von 76 ProbandInnen, etabliert, und mit 32 verblindeten ProbandInnen validiert. Mit dem CBA wurden in 23 der 23 NMDAR Enzephalitis PatientInnen spezifische Antikörper gefunden, doch in keiner der 85 Kontrollen (32 gesunde ProbandInnen und 53 PatientInnen mit anderen neurologischen Erkrankungen). Somit hatte der CBA eine Sensitivität und Spezifität von 100% (95% Konfidenzintervall (CI) 85.1-100.0 und 95.7-100.0). Mit dem FACS-basierten Test wurden spezifische Antikörper in 20 der 23 NMDAR Enzephalitis PatientInnen gefunden, und in keiner der 85 Kontrollen. Mit gleichbleibend hoher Spezifität (95% CI 95.7-100.0) hatte diese Testmethode daher eine niedrigere Sensitivität von 87% (95% CI 66.4-97.2) und zeigte eine hohe Variabilität. Beide Tests hatten eine hohe Konkordanz (kappa=0.943, p<0.0001) und Korrelation (r=0.697, p<0.0001). Mit dem zuverlässigeren CBA wurden bei einem Patienten mit NMDAR Enzephalitis (1/25; 4%) drei Jahre nach Erstmanifestation Hinweise auf Demyelinisierung zusammen mit Fortbestehen von NMDAR-Antikörpern gefunden. Des Weiteren wurden bei einer Patientin mit Multipler Sklerose (1/97; 1%) und NMDAR Enzephalitis-spezifischen Symptomen retrospektiv NMDAR Antikörper gefunden. Da die FACS-basierte Methode eine niedrigere Sensitivität, als auch eine hohe Variabilität aufweist, ist der CBA als zuverlässiger einzustufen. Zusammenfassend konnten wir zeigen, dass NMDAR Antikörper, wenngleich selten, mit demyelinisierenden Erkrankungen assoziiert sein können. Durch welche Mechanismen diese Antikörper zur Entstehung von Demyelinisierung beitragen, muss aber in weiteren Studien geklärt werden.N-methyl-D-aspartate receptor (NMDAR) encephalitis is a neurological autoimmune disease, diagnosed by a specific autoantibody against NMDAR. Among various antibody testing methods, cell-based assays (CBA) or immunohistochemistry on rat brain tissue have highest specificity and sensitivity. Nevertheless, results from antibody testing are controversially discussed. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Since a possible association of NMDAR antibodies with demyelinating diseases has been speculated, we further determined the frequency of NMDAR antibodies in cohorts of patients with various demyelinating diseases. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies andby Melanie RambergerZsfassung in dt. SpracheInnsbruck, Med. Univ., Diss., 2015OeBB(VLID)36986

CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1

Abstract Introduction: Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease. Methods: We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools. Results: Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope. Conclusions: A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients

Methodological Challenges in Protein Microarray and Immunohistochemistry for the Discovery of Novel Autoantibodies in Paediatric Acute Disseminated Encephalomyelitis

Acute disseminated encephalomyelitis (ADEM) is a rare autoimmune-mediated demyelinating disease affecting mainly children and young adults. Differentiation to multiple sclerosis is not always possible, due to overlapping clinical symptoms and recurrent and multiphasic forms. Until now, immunoglobulins reactive to myelin oligodendrocyte glycoprotein (MOG antibodies) have been found in a subset of patients with ADEM. However, there are still patients lacking autoantibodies, necessitating the identification of new autoantibodies as biomarkers in those patients. Therefore, we aimed to identify novel autoantibody targets in ADEM patients. Sixteen ADEM patients (11 seronegative, 5 seropositive for MOG antibodies) were analysed for potential new biomarkers, using a protein microarray and immunohistochemistry on rat brain tissue to identify antibodies against intracellular and surface neuronal and glial antigens. Nine candidate antigens were identified in the protein microarray analysis in at least two patients per group. Immunohistochemistry on rat brain tissue did not reveal new target antigens. Although no new autoantibody targets could be found here, future studies should aim to identify new biomarkers for therapeutic and prognostic purposes. The microarray analysis and immunohistochemistry methods used here have several limitations, which should be considered in future searches for biomarkers

Comparative Analysis of T-Cell Responses to Aquaporin-4 and Myelin Oligodendrocyte Glycoprotein in Inflammatory Demyelinating Central Nervous System Diseases

Autoantibodies against aquaporin-4 (AQP4-Ab) and myelin oligodendrocyte glycoprotein (MOG-Ab) are associated with rare central nervous system inflammatory demyelinating diseases like neuromyelitis optica spectrum disorders (NMOSD). Previous studies have shown that not only antibodies, but also autoreactive T-cell responses against AQP4 are present in NMOSD. However, no study has yet analyzed the presence of MOG reactive T-cells in patients with MOG antibodies. Therefore, we compared AQP4 and MOG specific peripheral T-cell response in individuals with AQP4-Ab (n = 8), MOG-Ab (n = 10), multiple sclerosis (MS, n = 8), and healthy controls (HC, n = 14). Peripheral blood mononuclear cell cultures were stimulated with eight AQP4 and nine MOG peptides selected from previous studies and a tetanus toxoid peptide mix as a positive control. Antigen-specific T-cell responses were assessed using the carboxyfluorescein diacetate succinimidyl ester proliferation assay and the detection of granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-ɤ and interleukin (IL)-4, IL-6, and IL-17A in cell culture supernatants. Additionally, human leukocyte antigen (HLA)-DQ and HLA-DR genotyping of all participants was performed. We classified a T-cell response as positive if proliferation (measured by a cell division index ≥3) was confirmed by the secretion of at least one cytokine. Reactivity against AQP4 peptides was observed in many groups, but the T-cell response against AQP4 p156-170 was present only in patients with AQP4-Ab (4/8, 50%) and absent in patients with MOG-Ab, MS and HC (corrected p = 0.02). This AQP4 p156-170 peptide specific T-cell response was significantly increased in participants with AQP4-Ab compared to those without [Odds ratio (OR) = 59.00, 95% confidence interval-CI 2.70-1,290.86]. Moreover, T-cell responses against at least one AQP4 peptide were also more frequent in participants with AQP4-Ab (OR = 11.45, 95% CI 1.24-106.05). We did not observe any significant differences for the other AQP4 peptides or any MOG peptide. AQP4-Ab were associated with HLA DQB1$^{*}$02 (OR = 5.71, 95% CI 1.09-30.07), DRB1$^{*}$01 (OR = 9.33, 95% CI 1.50-58.02) and DRB1$^{*}$03 (OR = 6.75, 95% CI = 1.19-38.41). Furthermore, HLA DRB1$^{*}$01 was also associated with the presence of AQP4 p156-170 reactive T-cells (OR = 31.67, 95% CI 1.30-772.98). To summarize, our findings suggest a role of AQP4-specific, but not MOG-specific T-cells, in NMOSD

Screening for pathogenic neuronal autoantibodies in serum and CSF of patients with first-episode psychosis

Patients with autoimmune encephalitides, especially those with antibodies to the N-methyl-D-aspartate receptor (NMDAR), often present with prominent psychosis and respond well to immunotherapies. Although most patients progress to develop various neurological symptoms, it has been hypothesised that a subgroup of patients with first-episode psychosis (FEP) suffer from a forme fruste of autoimmune encephalitis. Without accurate identification, this immunotherapy-responsive subgroup may be denied disease-modifying treatments. Thirty studies addressing aspects of this hypothesis were identified in a systematic review. Amongst other shortcomings, 15/30 reported no control group and only 6/30 determined cerebrospinal fluid (CSF) autoantibodies. To ourselves address these-and other-limitations, we investigated a prospectively ascertained clinically well-characterised cohort of 71 FEP patients without traditional neurological features, and 48 healthy controls. Serum and CSF were tested for autoantibodies against seven neuronal surface autoantigens using live cell-based assays. These identified 3/71 (4%) patient sera with weak binding to either contactin-associated protein-like 2, the NMDAR or glycine receptor versus no binding from 48 control samples (p = 0.28, Fisher's test). The three seropositive individuals showed no CSF autoantibodies and no differences from the autoantibody-negative patients in their clinical phenotypes, or across multiple parameters of peripheral and central inflammation. All individuals were negative for CSF NMDAR antibodies. In conclusion, formes frustes of autoimmune encephalitis are not prevalent among FEP patients admitted to psychiatric care. Our findings do not support screening for neuronal surface autoantibodies in unselected psychotic patients

No evidence for the involvement of leiomodin-1 antibodies in the pathogenesis of onchocerciasis-associated epilepsy

Nodding syndrome has been suggested to be triggered by neurotoxic leiomodin-1 auto-antibodies cross-reacting with Onchocerca volvulus. Here, we screened serum and CSF samples of persons with nodding syndrome and other forms of onchocerciasis-associated epilepsy (OAE) and African and European controls for leiomodin-1 antibodies by a cell-based assay (CBA) and Western blot (WB). These samples were also investigated for the presence of auto-antibodies cross-reacting with rat brain tissue by immunohistochemistry (IHC). Additionally, IHC was used to detect the leiomodin-1 protein in post-mortem brain samples of persons with OAE who died. Leiomodin-1 antibodies were detected by CBA in 6/52 (12%) and by WB in 23/54 (43%) persons with OAE compared to in 14/61 (23%) (p = 0.113) and 23/54 (43%) (p = 0.479) of controls without epilepsy. Multivariable exact logistic regression did not show an association between O. volvulus infection or epilepsy status and the presence of leiomodin-1. Leiomodin-1 antibodies were not detected in 12 CSF samples from persons with OAE or in 16 CSF samples from persons with acute-onset neurological conditions, as well as not being detected in serum from European controls. Moreover, the leiomodin-1 protein was only detected in capillary walls in post-mortem brain tissues and not in brain cells. IHC on rat brain slides with serum samples from persons with OAE or controls from persons with or without O. volvulus infection revealed no specific staining pattern. In conclusion, our data do not support OAE to be an autoimmune disorder caused by leiomodin-1 antibodies