Prospective open-label trial with rituximab in patients with chronic inflammatory demyelinating polyradiculoneuropathy not responding to conventional immune therapies.

BACKGROUND: To evaluate the efficacy of rituximab in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients not responding to conventional immune therapies. METHODS: An open-label, prospective exploratory study was conducted with intravenous rituximab on 17 CIDP patients who had not responded to at least two first-line therapies. The primary endpoint was to determine the proportion of patients who showed improvement 6 months after rituximab therapy. The percentage of responders to rituximab, along with a 95% CI, was reported and compared with the 30% response rate after other immunosuppressive drugs previously documented in the literature. RESULTS: 13 of the 17 treated patients (76.5%) showed improvement at 6 months (95% CI 50.1 to 93.2). Among the 14 patients who completed the 12-month follow-up (2 were lost to follow-up after showing improvement at months 8 and 10, and 1 deteriorated at 6 months), 13 (92.9%) demonstrated improvement at 12 months (95% CI 66.1 to 99.8). Nerve conduction parameters improved by at least 20% in two nerves in 6 out of 15 (40%) patients at 6 months and in 7 out of 13 (53.9%) at 12 months. None of the treated patients withdrew from the study due to side effects. There was a significant reduction of circulating CD19+ cells 15 days, 2, 6 and 12 months after treatment. CONCLUSION: Rituximab seems to be a safe therapy in most patients with CIDP not responding to conventional immune therapies. The high percentage of patients who improved in this study suggests a possible positive effect of rituximab which is worth investigating in future randomised controlled clinical trials. TRIAL REGISTRATION NUMBER: NCT05877040.

Excellent response to anti-CD38 therapy with daratumumab in a patient with severe refractory CANOMAD.

BACKGROUND: Intravenous immunoglobulin (IVIG) and rituximab are considered the first-line and second-line treatments for Chronic Ataxic Neuropathy and Ophthalmoplegia with IgM-paraprotein, cold Agglutinins, and anti-Disialosyl antibodies (CANOMAD), with an overall clinical response around 50%. New anti-CD38 daratumumab, targeting long-lived plasma cells, has been reported as a promising therapy for treatment-refractory antibody-mediated disorders. We report the first case of a severe refractory CANOMAD, successfully treated with daratumumab. METHODS: A patient in their 70s with severe relapsing CANOMAD, refractory to IVIG, steroids, rituximab and ibrutinib developed severe tetraparesis and respiratory failure. Plasma exchange (PE) improved motor and ventilatory function; however, after 6 weeks, patient remained PE dependent. Intravenous daratumumab was initiated at 16 mg/kg weekly for 3 weeks, every 2 weeks for the second and third month, and monthly afterwards. RESULTS: After 3 weeks of starting daratumumab, PE was discontinued and, since then, the patient evolved to complete recovery. Antidisialosyl antibody titres decreased after PE and remained stable during daratumumab. Serum neurofilament light-chain levels were elevated in the exacerbation phase and normalised after daratumumab. The patient remains in clinical remission under monthly daratumumab, 12 months after initiation. CONCLUSIONS: The first patient with aggressive treatment-refractory CANOMAD treated with daratumumab provides proof-of-principle evidence that daratumumab may be an effective treatment in IgM-related neuropathies.

A nationwide Guillain-Barré syndrome epidemiological study in Spain during the COVID-19 years.

BACKGROUND AND PURPOSE: The purpose was to perform a nationwide epidemiological study of Guillain-Barré syndrome (GBS) in Spain, analysing background incidences and seasonal variation and trying to identify incidence changes during the coronavirus disease 2019 (COVID-19) years. METHODS: This was an observational study collecting all GBS diagnoses from the National Epidemiological Surveillance Network collected by the Ministry of Health. Patients discharged with GBS as the main diagnosis and admitted during 2018-2021 were included. Data on the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections were obtained from the National Epidemiology Centre. RESULTS: In total, 3147 cases were included, 832 in 2018, 861 in 2019, 670 in 2020 and 784 in 2021. Nationwide hospital incidence was 1.78 in 2018, 1.71 in 2019, 1.41 in 2020 and 1.66 in 2021, with an increased frequency in males, the elderly population and in the winter season. Eleven per cent of GBS patients needed ventilatory support. GBS and SARS-CoV-2 incidences did not correlate with one another (r = -0.29, p = 0.36). GBS incidence decreased during 2020 and during the COVID-19 lockdown period in comparison to the same months of 2018-2019. CONCLUSIONS: The incidence of GBS in Spain is similar to that of other countries. Despite prior reports describing a significant increase in COVID-19-associated GBS in Spain, a significant drop of GBS incidence during the SARS-CoV-2 pandemic was detected, probably due to prevention measures.

Clinical relevance of distinguishing autoimmune nodopathies from CIDP: longitudinal assessment in a large cohort.

BACKGROUND: The aim of this study was to determine treatment response and whether it is associated with antibody titre change in patients with autoimmune nodopathy (AN) previously diagnosed as chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and to compare clinical features and treatment response between AN and CIDP. METHODS: Serum IgG antibodies to neurofascin-155 (NF155), contactin-1 (CNTN1) and contactin-associated protein 1 (CASPR1) were detected with cell-based assays in patients diagnosed with CIDP. Clinical improvement was determined using the modified Rankin scale, need for alternative and/or additional treatments and assessment of the treating neurologist. RESULTS: We studied 401 patients diagnosed with CIDP and identified 21 patients with AN (10 anti-NF155, 6 anti-CNTN1, 4 anti-CASPR1 and 1 anti-NF155/anti-CASPR1 double positive). In patients with AN ataxia (68% vs 28%, p=0.001), cranial nerve involvement (34% vs 11%, p=0.012) and autonomic symptoms (47% vs 22%, p=0.025) were more frequently reported; patients with AN improved less often after intravenous immunoglobulin treatment (39% vs 80%, p=0.002) and required additional/alternative treatments more frequently (84% vs 34%, p<0.001), compared with patients with CIDP. Antibody titres decreased or became negative in patients improving on treatment. Treatment withdrawal was associated with a titre increase and clinical deterioration in four patients. CONCLUSIONS: Distinguishing CIDP from AN is important, as patients with AN need a different treatment approach. Improvement and relapses were associated with changes in antibody titres, supporting the pathogenicity of these antibodies.

The autoimmune vulnerability of the node of Ranvier.

The nodes of Ranvier (NoR) are essential domains for nerve conduction and their disruption plays a key role in the pathophysiology of immune-mediated neuropathies. Our understanding of the specialized nodal regions and the immune mechanisms that affect them is growing and has led to the update of peripheral neuropathy classification to include the autoimmune nodopathies, defined by the site of the autoimmune attack. Autoantibodies directed against molecules of the nodal region (as neurofascin-140/186, neurofascin-155, contactin-1, contactin-associated protein 1, contactin-associated protein 2, gangliosides, LGI4, or myelin-associated glycoprotein), macrophage-induced paranodal demyelination, and phenotypic changes of the nodal domains of Schwann cells have been identified as key mechanisms in the pathogenesis of the autoimmune neuropathies. This review explores the current knowledge of the autoimmune vulnerability of the NoR, including the underlying mechanisms leading to dysfunction in the diverse autoimmune disorders.

Autoimmune nodopathies, an emerging diagnostic category.

PURPOSE OF REVIEW: In the last decade, antibodies targeting cell adhesion molecules of the node of Ranvier were described in patients with autoimmune neuropathies. These nodal/paranodal antibodies associate with specific clinicopathological features that are different from classical chronic inflammatory demyelinating polyneuropathy (CIDP). In this review, we will summarize recent findings establishing autoimmune nodopathies (AN) as a new category of autoimmune neuropathies. RECENT FINDINGS: AN include anti-contactin 1, anti-contactin-associated protein 1, anti-neurofascin 155 and anti-pan-neurofascin antibody-mediated neuropathies. Their clinical spectrum includes acute, subacute or chronic onset sensory-motor neuropathies mimicking Guillain-Barré syndrome (GBS) and CIDP, although they differ in their response to standard therapy with intravenous immunoglobulin (IVIG). Neurophysiologically they overlap with acquired demyelinating neuropathies, but ultrastructural studies and animal models demonstrated antibody-mediated pathology restricted to the node of Ranvier. Anti-contactin1 and anti-pan-neurofascin also associate with nephrotic syndrome. Nodal/paranodal antibodies are predominantly of the immunoglobulin (IgG)4 subclass during the chronic phase of the disease, but complement-fixing IgG3 antibodies are detected during the early phase and associate with aggressive onset and IVIG response. Nodal/paranodal antibodies testing is key in the diagnosis of AN. SUMMARY: AN have emerged as a new diagnostic category pathologically different from acquired demyelinating neuropathies. Clinically they overlap with GBS and CIDP although they associate with specific clinical features that should lead to clinical suspicion. Nodal/paranodal antibodies are key effector mechanisms of disease and good diagnostic and disease-monitoring biomarkers in AN.

Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies.

Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss.

Antibodies to the Caspr1/contactin-1 complex in chronic inflammatory demyelinating polyradiculoneuropathy.

Previous studies have described the clinical, serological and pathological features of patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and antibodies directed against the paranodal proteins neurofascin-155, contactin-1 (CNTN1), contactin-associated protein-1 (Caspr1), or nodal forms of neurofascin. Such antibodies are useful for diagnosis and potentially treatment selection. However, antibodies targeting Caspr1 only or the Caspr1/CNTN1 complex have been reported in few patients with CIDP. Moreover, it is unclear if these patients belong to the same pathophysiological subgroup. Using cell-based assays in routine clinical testing, we identified sera from patients with CIDP showing strong membrane reactivity when both CNTN1 and Caspr1 were co-transfected (but not when CNTN1 was transfected alone). Fifteen patients (10 male; aged between 40 and 75) with antibodies targeting Caspr1/CNTN1 co-transfected cells were enrolled for characterization. The prevalence of anti-Caspr1/CNTN1 antibodies was 1.9% (1/52) in the Sant Pau CIDP cohort, and 4.3% (1/23) in a German cohort of acute-onset CIDP. All patients fulfilled European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) definite diagnostic criteria for CIDP. Seven (47%) were initially diagnosed with Guillain-Barré syndrome due to an acute-subacute onset. Six (40%) patients had cranial nerve involvement, eight (53%) reported neuropathic pain and 12 (80%) ataxia. Axonal involvement and acute denervation were frequent in electrophysiological studies. Complete response to intravenous immunoglobulin was not observed, while most (90%) responded well to rituximab. Enzyme-linked immunosorbent assay (ELISA) and teased nerve fibre immunohistochemistry confirmed reactivity against the paranodal Caspr1/CNTN1 complex. Weaker reactivity against Caspr1 transfected alone was also detected in 10/15 (67%). Sera from 13 of these patients were available for testing by ELISA. All 13 samples reacted against Caspr1 by ELISA and this reactivity was enhanced when CNTN1 was added to the Caspr1 ELISA. IgG subclasses were also investigated by ELISA. IgG4 was the predominant subclass in 10 patients, while IgG3 was predominant in other three patients. In conclusion, patients with antibodies to the Caspr1/CNTN1 complex display similar serological and clinical features and constitute a single subgroup within the CIDP syndrome. These antibodies likely target Caspr1 primarily and are detected with Caspr1-only ELISA, but reactivity is optimal when CNTN1 is added to Caspr1 in cell-based assays and ELISA.

Autoantibody screening in Guillain-Barré syndrome.

BACKGROUND: Guillain-Barré syndrome (GBS) is an acute inflammatory neuropathy with a heterogeneous presentation. Although some evidences support the role of autoantibodies in its pathogenesis, the target antigens remain unknown in a substantial proportion of GBS patients. The objective of this study is to screen for autoantibodies targeting peripheral nerve components in Guillain-Barré syndrome. METHODS: Autoantibody screening was performed in serum samples from all GBS patients included in the International GBS Outcome study by 11 different Spanish centres. The screening included testing for anti-ganglioside antibodies, anti-nodo/paranodal antibodies, immunocytochemistry on neuroblastoma-derived human motor neurons and murine dorsal root ganglia (DRG) neurons, and immunohistochemistry on monkey peripheral nerve sections. We analysed the staining patterns of patients and controls. The prognostic value of anti-ganglioside antibodies was also analysed. RESULTS: None of the GBS patients (n = 100) reacted against the nodo/paranodal proteins tested, and 61 (61%) were positive for, at least, one anti-ganglioside antibody. GBS sera reacted strongly against DRG neurons more frequently than controls both with IgG (6% vs 0%; p = 0.03) and IgM (11% vs 2.2%; p = 0.02) immunodetection. No differences were observed in the proportion of patients reacting against neuroblastoma-derived human motor neurons. Reactivity against monkey nerve tissue was frequently detected both in patients and controls, but specific patterns were only detected in GBS patients: IgG from 13 (13%) patients reacted strongly against Schwann cells. Finally, we confirmed that IgG anti-GM1 antibodies are associated with poorer outcomes independently of other known prognostic factors. CONCLUSION: Our study confirms that (1) GBS patients display a heterogeneous repertoire of autoantibodies targeting nerve cells and structures; (2) gangliosides are the most frequent antigens in GBS patients and have a prognostic value; (3) further antigen-discovery experiments may elucidate other potential antigens in GBS.

Serum neurofilament light chain predicts long-term prognosis in Guillain-Barré syndrome patients.

OBJECTIVE: To study baseline serum neurofilament light chain (sNfL) levels as a prognostic biomarker in Guillain-Barré syndrome (GBS). METHODS: We measured NfL in serum (98 samples) and cerebrospinal fluid (CSF) (24 samples) of patients with GBS prospectively included in the International GBS Outcome Study (IGOS) in Spain using single-molecule array (SiMoA) and compared them with 53 healthy controls (HCs). We performed multivariable regression to analyse the association between sNfL levels and functional outcome at 1 year. RESULTS: Patients with GBS had higher NfL levels than HC in serum (55.49 pg/mL vs 9.83 pg/mL, p<0.0001) and CSF (1308.5 pg/mL vs 440.24 pg/mL, p=0.034). Patients with preceding diarrhoea had higher sNfL than patients with respiratory symptoms or no preceding infection (134.90 pg/mL vs 47.86 pg/mL vs 38.02 pg/mL, p=0.016). sNfL levels correlated with Guillain-Barré Syndrome Disability Score and Inflammatory Rasch-built Overall Disability Scale (I-RODS) at every timepoint. Patients with pure motor variant and Miller Fisher syndrome showed higher sNfL levels than patients with sensorimotor GBS (162.18 pg/mL vs 95.50 pg/mL vs 38.02 pg/mL, p=0.025). Patients with acute motor axonal neuropathy cute motor axonal neuropathy had higher sNfL levels than other variants (190.55 pg/mL vs 46.79 pg/mL, p=0.013). sNfL returned to normal levels at 1 year. High baseline sNfL levels were associated with inability to run (OR=1.65, 95% CI 1.14 to 2.40, p=0.009) and lower I-RODS (ß -2.60, 95% CI -4.66 to -0.54, p=0.014) at 1 year. Cut-off points predicting clinically relevant outcomes at 1 year with high specificity were calculated: inability to walk independently (>319 pg/mL), inability to run (>248 pg/mL) and ability to run (<34 pg/mL). CONCLUSION: Baseline sNfL levels are increased in patients with GBS, are associated with disease severity and axonal variants and have an independent prognostic value in patients with GBS.

Proteasome inhibitors reduce thrombospondin-1 release in human dysferlin-deficient myotubes.

BACKGROUND: Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44. METHODS: To assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p ≤ 0.05 was considered statistically significant. RESULTS: Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury. CONCLUSIONS: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

Membrane Proteome-Wide Screening of Autoantibodies in CIDP Using Human Cell Microarray Technology.

BACKGROUND AND OBJECTIVES: Autoantibody discovery in complex autoimmune diseases is challenging. Diverse successful antigen identification strategies are available, but, so far, have often been unsuccessful, especially in the discovery of protein antigens in which conformational and post-translational modification are critical. Our study assesses the utility of a human membrane and secreted protein microarray technology to detect autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: A cell microarray consisting of human embryonic kidney-293 cells expressing >5,000 human proteins was used. First, a validation step was performed with 4 serum samples from patients with autoimmune nodopathy (AN) to assess the ability of this technology to detect circulating known autoantibodies. The ability of the cell microarray technology to discover novel IgG autoantibodies was assessed incubating the array with 8 CIDP serum samples. Identified autoantibodies were subsequently validated using cell-based assays (CBAs), ELISA, and/or tissue immunohistochemistry and analyzed in a cohort of CIDP and AN (n = 96) and control (n = 100) samples. RESULTS: Serum anti-contactin-1 and anti-neurofascin-155 were detected by the human cell microarray technology. Nine potentially relevant antigens were found in patients with CIDP without other detectable antibodies; confirmation was possible in six of them: ephrin type-A receptor 7 (EPHA7); potassium-transporting ATPase alpha chain 1 and subunit beta (ATP4A/4B); leukemia-inhibitory factor (LIF); and interferon lambda 1, 2, and 3 (IFNL1, IFNL2, IFNL3). Anti-ATP4A/4B and anti-EPHA7 antibodies were detected in patients and controls and considered unrelated to CIDP. Both anti-LIF and anti-IFNL antibodies were found in the same 2 patients and were not detected in any control. Both patients showed the same staining pattern against myelinating fibers of peripheral nerve tissue and of myelinating neuron-Schwann cell cocultures. Clinically relevant correlations could not be established for anti-LIF and anti-IFNL3 antibodies. DISCUSSION: Our work demonstrates the utility of human cell microarray technology to detect known and discover unknown autoantibodies in human serum samples. Despite potential CIDP-associated autoantibodies (anti-LIF and anti-IFNL3) being identified, their clinical and pathogenic relevance needs to be elucidated in bigger cohorts.

Absence of Pathogenic Mutations and Strong Association With HLA-DRB1*11:01 in Statin-Naïve Early-Onset Anti-HMGCR Necrotizing Myopathy.

BACKGROUND AND OBJECTIVES: Immune-mediated necrotizing myopathy (IMNM) caused by antibodies against 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is an inflammatory myopathy that has been epidemiologically correlated with previous statin exposure. We characterized in detail a series of 11 young statin-naïve patients experiencing a chronic disease course mimicking a limb-girdle muscular dystrophy. With the hypothesis that HMGCR upregulation may increase immunogenicity and trigger the production of autoantibodies, our aim was to expand pathophysiologic knowledge of this distinct phenotype. METHODS: Clinical and epidemiologic data, autoantibody titers, creatine kinase (CK) levels, response to treatment, muscle imaging, and muscle biopsies were assessed. HMGCR expression in patients' muscle was assessed by incubating sections of affected patients with purified anti-HMGCR+ serum. Whole-exome sequencing (WES) with a special focus on cholesterol biosynthesis-related genes and high-resolution human leukocyte antigen (HLA) typing were performed. RESULTS: Patients, aged 3-25 years and mostly female (90.9%), presented with subacute proximal weakness progressing over many years and high CK levels (>1,000 U/L). Diagnostic delay ranged from 3 to 27 years. WES did not reveal any pathogenic variants. HLA-DRB1*11:01 carrier frequency was 60%, a significantly higher proportion than in the control population. No upregulation or mislocalization of the enzyme in statin-exposed or statin-naïve anti-HMGCR+ patients was observed, compared with controls. DISCUSSION: WES of a cohort of patients with dystrophy-like anti-HMGCR IMNM did not reveal any common rare variants of any gene, including cholesterol biosynthesis-related genes. HLA analysis showed a strong association with HLA-DRB1*11:01, previously mostly described in statin-exposed adult patients; consequently, a common immunogenic predisposition should be suspected, irrespective of statin exposure. Moreover, we were unable to conclusively demonstrate muscle upregulation/mislocalization of HMGCR in IMNM, whether or not driven by statins.

Effect of monovalency on anti-contactin-1 IgG4.

Introduction: Autoimmune nodopathies (AN) have been diagnosed in a subset of patients fulfilling criteria for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who display no or poor response to intravenous immunoglobulins. Biomarkers of AN are autoantibodies, mainly IgG4, directed against the ternary paranodal complex composed by neurofascin-155, contactin-1 (CNTN1), and Contactin-associated-protein-1 (CASPR1) or against the nodal isoforms of neurofascin. IgG4 can undergo a Fab-arm exchange (FAE) which results in functionally monovalent antibody. This phenomenon differentially affects the pathogenicity of IgG4 depending on the target of autoantibodies. Here, we have evaluated this issue by examining the impact of valency on anti-CNTN1 IgG4 which induces paranodal destruction through a function blocking activity. Methods: Sera were obtained from 20 patients with AN associated with anti-CNTN1 antibodies. The proportion of monospecific/bispecific anti-CNTN1 antibodies was estimated in each patient by ELISA by examining the ability of serum antibodies to cross-link untagged CNTN1 with biotinylated CNTN1. To determine the impact of monovalency, anti-CNTN1 IgG4 were enzymatically digested into monovalent Fab and tested in vitro on cell aggregation assay. Also, intraneural injections were performed to determine whether monovalent Fab and native IgG4 may penetrate paranode, and antibody infiltration was monitored 1- and 3-days post injection. Results and discussion: We found that the percentage of monospecific antibodies were lower than 5% in 14 out of 20 patients (70%), suggesting that IgG4 have undergone extensive FAE in situ. The levels of monospecific antibodies correlated with the titers of anti-CNTN1 antibodies. However, no correlation was found with clinical severity, and patients with low or high percentage of monospecific antibodies similarly showed a severe phenotype. Native anti-CNTN1 IgG4 were shown to inhibit the interaction between cells expressing CNTN1/CASPR1 and cells expressing neurofascin-155 using an in vitro aggregation assay. Similarly, monovalent Fab significantly inhibited the interaction between CNTN1/CASPR1 and neurofascin-155. Intraneural injections of Fab and native anti-CNTN1 IgG4 indicated that both mono- and bivalent anti-CNTN1 IgG4 potently penetrated the paranodal regions and completely invaded this region by day 3. Altogether, these data indicate anti-CNTN1 IgG4 are mostly bispecific in patients, and that functionally monovalent anti-CNTN1 antibodies have the pathogenic potency to alter paranode.

Neuroinflammation-Related Proteins NOD2 and Spp1 Are Abnormally Upregulated in Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology and poorly understood pathophysiology. There is no specific biomarker either for diagnosis or prognosis. The aim of our study was to investigate differentially expressed proteins in the CSF and serum from patients with ALS to determine their role in the disease process and evaluate their utility as diagnostic or prognostic biomarkers. METHODS: We performed mass spectrometry in the CSF from 3 patients with ALS and 3 healthy controls (HCs). The results were compared with motor cortex dysregulated transcripts obtained from 11patients with sporadic ALS and 8 HCs. Candidate proteins were tested using ELISA in the serum of 123 patients with ALS, 30 patients with Alzheimer disease (AD), 28 patients with frontotemporal dementia (FTD), and 102 HCs. Patients with ALS, AD, and FTD were prospectively recruited from January 2003 to December 2020. A group of age-matched HCs was randomly selected from the Sant Pau Initiative on Neurodegeneration cohort of the Sant Pau Memory Unit. RESULTS: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and osteopontin (Spp1) were differentially expressed in the CSF and the motor cortex transcriptome of patients with ALS compared with that in HCs (p < 0.05). NOD2 and Spp1 levels were significantly higher in sera from patients with ALS than in HCs (p < 0.001). Receiver operating characteristic analysis showed an area under the curve of 0.63 for NOD2 and 0.81 for Spp1. NOD2 levels were significantly lower in patients with AD and FTD than in patients with ALS (p < 0.0001), but we found no significant differences in Spp1 levels between patients with ALS, AD (p = 0.51), and FTD (p = 0.42). We found a negative correlation between Spp1 levels and ALS functional rating scale (r = -0.24, p = 0.009). DISCUSSION: Our discovery-based approach identified NOD2 as a novel biomarker in ALS and adds evidence to the contribution of Spp1 in the disease process. Both proteins are involved in innate immunity and autophagy and are increased in the serum from patients with ALS. Our data support a relevant role of neuroinflammation in the pathophysiology of the disease and may identify targets for disease-modifying treatments in ALS. Further longitudinal studies should investigate the diagnostic and prognostic value of NOD2 and Spp1 in clinical practice.

Guillain-Barré Syndrome Following Zika Virus Infection Is Associated With a Diverse Spectrum of Peripheral Nerve Reactive Antibodies.

BACKGROUND AND OBJECTIVES: Recent outbreaks of Zika virus (ZIKV) in South and Central America have highlighted significant neurologic side effects. Concurrence with the inflammatory neuropathy Guillain-Barré syndrome (GBS) is observed in 1:4,000 ZIKV cases. Whether the neurologic symptoms of ZIKV infection are immune mediated is unclear. We used rodent and human live cellular models to screen for anti-peripheral nerve reactive IgG and IgM autoantibodies in the sera of patients with ZIKV with and without GBS. METHODS: In this study, 52 patients with ZIKV-GBS were compared with 134 ZIKV-infected patients without GBS and 91 non-ZIKV controls. Positive sera were taken forward for target identification by immunoprecipitation and mass spectrometry, and candidate antigens were validated by ELISA and cell-based assays. Autoantibody reactions against glycolipid antigens were also screened on an array. RESULTS: Overall, IgG antibody reactivities to rat Schwann cells (SCs) (6.5%) and myelinated cocultures (9.6%) were significantly higher, albeit infrequent, in the ZIKV-GBS group compared with all controls. IgM antibody immunoreactivity to dorsal root ganglia neurones (32.3%) and SCs (19.4%) was more frequently observed in the ZIKV-GBS group compared with other controls, whereas IgM reactivity to cocultures was as common in ZIKV and non-ZIKV sera. Strong axonal-binding ZIKV-GBS serum IgG antibodies from 1 patient were confirmed to react with neurofascin 155 and 186. Serum from a ZIKV-infected patient without GBS displayed strong myelin-binding and putative antilipid antigen reaction characteristics. There was, however, no significant association of ZIKV-GBS with any known antiglycolipid antibodies. DISCUSSION: Autoantibody responses in ZIKV-GBS target heterogeneous peripheral nerve antigens suggesting heterogeneity of the humoral immune response despite a common prodromal infection.

Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis.

Multiple sclerosis is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 or neurofascin-155 have been previously described in multiple sclerosis patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against neurofascin-155, anoctamin-2 and flotillin-1/2 complex in multiple sclerosis. Serum (n = 252) and CSF (n = 50) samples from 282 multiple sclerosis patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-flotillin-1/2, anti-anoctamin-2 and anti-neurofascin-155 antibodies were tested by cell-based assays using transfected cells. We identified six multiple sclerosis patients with antibodies against the flotillin-1/2 complex (2.1%) and one multiple sclerosis patient with antibodies against anoctamin-2 (0.35%). All multiple sclerosis patients were negative for anti-neurofascin-155 antibodies. Three of the anti-flotillin-1/2 positive patients showed anti-flotillin-1/2 positivity in other serum samples extracted at different moments of their disease. Immunoglobulin G subclasses of anti-flotillin-1/2 antibodies were predominantly one and three. We confirm that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis.

mRNA COVID-19 Vaccination Does Not Exacerbate Symptoms or Trigger Neural Antibody Responses in Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: In people with multiple sclerosis (pwMS), concern for potential disease exacerbation or triggering of other autoimmune disorders contributes to vaccine hesitancy. We assessed the humoral and T-cell responses to SARS-CoV-2 after mRNA vaccination, changes in disease activity, and development of antibodies against central or peripheral nervous system antigens. METHODS: This was a prospective 1-year longitudinal observational study of pwMS and a control group of patients with other inflammatory neurologic disorders (OIND) who received an mRNA vaccine. Blood samples were obtained before the first dose (T1), 1 month after the first dose (T2), 1 month after the second dose (T3), and 6 (T4), 9 (T5), and 12 (T6) months after the first dose. Patients were assessed for the immune-specific response, annualized relapse rate (ARR), and antibodies to onconeuronal, neural surface, glial, ganglioside, and nodo-paranodal antigens. RESULTS: Among 454 patients studied, 390 had MS (22 adolescents) and 64 OIND; the mean (SD) age was 44 (14) years; 315 (69%) were female; and 392 (87%) were on disease-modifying therapies. Antibodies to the receptor-binding domain were detected in 367 (86%) patients at T3 and 276 (83%) at T4. After a third dose, only 13 (22%) of 60 seronegative patients seroconverted, and 255 (92%) remained seropositive at T6. Cellular responses were present in 381 (93%) patients at T3 and in 235 (91%) patients at T6 including all those receiving anti-CD20 therapies and in 79% of patients receiving fingolimod. At T3 (429 patients) or T6 (395 patients), none of the patients had developed CNS autoantibodies. Seven patients had neural antibodies that were already present before immunization (3 adult patients with MS had MOG-IgG, 2 with MG and 1 with MS had neuronal cell surface antibodies [unknown antigen], and 1 with MS had myelin antibody reactivity [unknown antigen]. Similarly, no antibodies against PNS antigens were identified at T3 (427 patients). ARR was lower in MS and not significantly different in patients with OIND. Although 182 (40%) patients developed SARS-CoV-2 infection, no cases of severe COVID-19 or serious adverse events occurred. DISCUSSION: In this study, mRNA COVID-19 vaccination was safe and did not exacerbate the autoimmune disease nor triggered neural autoantibodies or immune-mediated neurologic disorders. The outcome of patients who developed SARS-CoV-2 infection was favorable.

IgG4 Valency Modulates the Pathogenicity of Anti-Neurofascin-155 IgG4 in Autoimmune Nodopathy.

BACKGROUND AND OBJECTIVES: IgG4 autoantibodies to neurofascin-155 (Nfasc155) are associated with a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (CIDP), currently named autoimmune nodopathy. We previously demonstrated that those antibodies alter conduction along myelinated axons by inducing Nfasc155 depletion and paranode destruction. In blood, IgG4 have the potency to exchange their moiety with other unrelated IgG4 through a process called Fab-arm exchange (FAE). This process results in functionally monovalent antibodies and may affect the pathogenicity of autoantibodies. Here, we examined this issue and whether FAE is beneficial or detrimental for Nfasc155 autoimmune nodopathy. METHODS: The bivalency and monospecificity of anti-Nfasc155 were examined by sandwich ELISA in 10 reactive patients, 10 unreactive CIDP patients, and 10 healthy controls. FAE was induced in vitro using reduced glutathione and unreactive IgG4, and the ratio of the κ:λ light chain was monitored. To determine the pathogenic potential of bivalent anti-Nfasc155 IgG4, autoantibodies derived from patients were enzymatically cleaved into monovalent Fab and bivalent F(ab')2 or swapped with unreactive IgG4 and then were injected in neonatal animals. RESULTS: Monospecific bivalent IgG4 against Nfasc155 were detected in the serum of all reactive patients, indicating that a fraction of IgG4 have not undergone FAE in situ. These IgG4 were, nonetheless, capable of engaging into FAE with unreactive IgG4 in vitro, and this decreased the levels of monospecific antibodies and modulated the ratio of the κ:λ light chain. When injected in animals, monovalent anti-Nfasc155 Fab did not alter the formation of paranodes; by contrast, both native anti-Nfasc155 IgG4 and F(ab')2 fragments strongly impaired paranode formation. The promotion of FAE with unreactive IgG4 also strongly diminished the pathogenic potential of anti-Nfasc155 IgG4 in animals and decreased IgG4 clustering on Schwann cells. DISCUSSION: Our findings demonstrate that monospecific and bivalent anti-Nfasc155 IgG4 are detected in patients and that those autoantibodies are the pathogenic ones. The transformation of anti-Nfasc155 IgG4 into monovalent Fab or functionally monovalent IgG4 through FAE strongly decreases paranodal alterations. Bivalency thus appears crucial for Nfasc155 clustering and paranode destruction.

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice.

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca-/- mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca-/- mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca-/- mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca-/- mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy.