Neuromyelitis optica and neuromyelitis optica spectrum disorders have been recently associated with the disease-specific autoantibody aquaporin-4, thought to be pathogenic. Identifying this antibody ...has allowed the clinical phenotype to be broadened. It is clear that some patients with similar clinical features do not have this antibody and may have a different condition with different outcomes and prognosis. Previous clinical neuromyelitis optica and neuromyelitis optica spectrum disorder studies have included such patients. We investigated clinical outcomes and prognostic characteristics of 106 aquaporin-4 antibody-seropositive patients from the UK and Japan. We looked at predictors of disability outcomes, namely visual disability (permanent bilateral visual loss with visual acuity of <6/36 in the best eye), motor disability (permanent inability to walk further than 100 m unaided), wheelchair dependence and mortality. Data were collected largely retrospectively through review of case records. After median disease duration of 75 months, 18% had developed permanent bilateral visual disability, 34% permanent motor disability, 23% had become wheelchair dependent and 9% had died. Age at disease onset appeared to be an important predictor of disability type. Young-onset patients in the UK, but not the Japanese cohort, commonly presenting with optic neuritis, had a high risk of visual disability while older patients in both cohorts had a high risk of motor disability, regardless of their onset symptom. Genetic factors also appeared important. The UK cohort seemed to have more severe disease than the Japanese cohort, with more severe onset attacks, a higher relapse frequency and greater disability at follow-up, despite earlier immunosuppression. Moreover, within the UK cohort, there were important differences between ethnic groups, with Afro-Caribbean patients having a younger age at disease onset, more brain and multifocal attacks and higher likelihood of visual disability than Caucasian patients. Thus, age at disease onset and genetic factors are both likely to be important in determining clinical outcomes in aquaporin-4 disease. This has important implications for interpreting clinical neuromyelitis optica and neuromyelitis optica spectrum disorder studies, since clinical features and outcomes appear not to be generic across populations and may need to be tailored to individual groups. These factors need to be explored further in future prospective neuromyelitis optica and neuromyelitis optica spectrum disorder studies.
Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the ...dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5−7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.
Aquaporin-4 antibodies are pathogenic in neuromyelitis optica spectrum disorders (NMOSD). Wilson et al. show that circulating memory and naïve B cells from patients can differentiate to produce ...aquaporin-4 antibodies under conditions chosen to mimic aspects of NMOSD. This platform should direct rationale immunotherapy in NMOSD and other antibody-mediated conditions.
Abstract
Autoantibodies to aquaporin-4 (AQP4) are pathogenic in neuromyelitis optica spectrum disorder (NMOSD). However, it is not known which B cells are the major contributors to circulating AQP4 antibodies nor which conditions promote their generation. Our experiments showed CD19+CD27++CD38++ circulating ex vivo antibody-secreting cells did not produce AQP4 antibodies under several culture conditions. To question whether other cells in circulation were capable of AQP4 antibody production, B cells were differentiated into antibody-secreting cells in vitro. Unfractionated peripheral blood mononuclear cells, isolated from 12 patients with NMOSD and a wide range of serum AQP4 antibody levels (91-26 610 units), were cultured with factors that mimicked established associations of NMOSD including T cell help, concurrent infections and cytokines reported to be elevated in NMOSD. Overall, the in vitro generation of CD19+CD27++CD38++ cells across several culture conditions correlated closely with the total IgG secreted (P < 0.0001, r = 0.71), but not the amount of AQP4 antibody. AQP4 antibody production was enhanced by CD40-ligand (P = 0.005), and by interleukin-2 plus toll-like receptor stimulation versus interleukin-21-predominant conditions (P < 0.0001), and did not require antigen. Across NMOSD patients, this in vitro generation of AQP4 antibodies correlated well with serum AQP4 antibody levels (P = 0.0023, r = 0.81). To understand how early within B cell lineages this AQP4 specificity was generated, purified B cell subsets were activated under these optimized conditions. Naïve pre-germinal centre B cells (CD19+CD27−IgD+) differentiated to secrete AQP4 antibodies as frequently as post-germinal centre cells (CD19+CD27+). Taken together, these human cell-culture experiments demonstrate that preformed B cells, rather than ex vivo circulating antibody-secreting cells, possess AQP4 reactivity. Their differentiation and AQP4 antibody secretion is preferentially driven by select cytokines and these cells may make the dominant contribution to serum AQP4 antibodies. Furthermore, as AQP4-specific B cells can derive from likely autoreactive naïve populations an early, pre-germinal centre loss of immunological tolerance appears present in some patients with NMOSD. This study has implications for understanding mechanisms of disease perpetuation and for rational choice of immunotherapies in NMOSD. Furthermore, the in vitro model presents an opportunity to apply condition-specific approaches to patients with NMOSD and may be a paradigm to study other antibody-mediated diseases.
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IMPORTANCE Aquaporin 4 antibody (AQP4-Ab)–negative patients with longitudinally extensive transverse myelitis (LETM) behave differently from those with AQP4-Ab. Aquaporin 4 antibody–negative ...neuromyelitis optica (NMO) is rare when good assays are used. OBJECTIVE To assess if AQP4-Ab–negative patients with LETM share similar disease characteristics with AQP4-Ab–positive patients or whether they have distinct features and alternative diagnoses. DESIGN We collated clinical and paraclinical data on patients with LETM identified through the Oxford NMO clinical database. Aquaporin 4 antibodies were tested using 2 sensitive assays. We describe the features of patients with LETM, compare findings between patients with and without AQP4-Ab, and describe alternative diagnoses in AQP4-Ab–negative patients. SETTING Single specialist UK center for NMO. PARTICIPANTS Seventy-six adult patients with LETM. MAIN OUTCOMES AND MEASURES Comparison of clinical and paraclinical data. RESULTS Fifty-eight percent of patients were AQP4-Ab positive. Alternative diagnoses could usually be identified in AQP4-Ab–negative patients, including those fulfilling NMO diagnostic criteria. Only 6.5% of patients had “true” seronegative NMO and 6.5% had idiopathic LETM. There were some important differences between AQP4-Ab–positive and –negative cases, including older onset age, higher proportion of females, lower incidence of simultaneous optic neuritis, lower frequency of conus involvement, and higher prevalence of coexisting autoimmune disorders in AQP4-Ab–positive cases. Attack severity and degree of recovery were similar in the 2 groups. CONCLUSIONS AND RELEVANCE Patients with LETM without AQP4-Ab include a number of different diagnostic categories and it is not surprising therefore that they show important differences compared with AQP4-Ab–positive patients, even when considering only those fulfilling current NMO diagnostic criteria. Thus, we suggest that diagnoses such as myelin-oligodendrocyte glycoprotein antibody disease, multiple sclerosis, acute disseminated encephalomyelitis, and postinfectious disorders should be exclusions in the NMO diagnostic criteria and AQP4-Ab–positive and antibody–negative NMO/NMO spectrum disorder cohorts should be analyzed separately.
Myelin oligodendrocyte glycoprotein (MOG) is a unique CNS-specific mammalian protein that is expressed on the surface of compact myelin and oligodendrocyte cell bodies. MOG is an accessible target ...for autoantibodies, associated with immune-mediated demyelination in the central nervous system. The identification of MOG reactive immunoglobulin G antibodies (MOG-IgG) helps to distinguish a subgroup of patients from multiple sclerosis and other CNS disorders, reducing the risk of clinical misdiagnosis. The development of the cell-based assays (CBA) improved the detection of clinically meaningful MOG-IgG binding to conformational MOG expressed in the cell membrane surface. In this review, we describe factors that impact on the results of CBA, such as MOG conformation, protein glycosylation, addition of fluorescent tags, serum dilution, secondary antibodies, and data interpretation.
SignificanceBy studying paired blood and deep cervical lymph node samples from patients with neuromyelitis optica spectrum disorders, our data provide evidence for a germinal center-based generation ...of aquaporin-4 antibodies. Frequent serum aquaporin-4 immunoglobulin Ms (IgMs) and shifts in IgG subclasses were observed alongside preferential synthesis of aquaporin-4 IgGs and aquaporin-4-reactive B cells within lymph nodes. Both intranodal synthesis of aquaporin-4 antibodies and intranodal aquaporin-4-reactive B cells were robustly eliminated with rituximab administration. This study systematically explores lymph nodes that drain the central nervous system (CNS) in patients with CNS autoimmunity and offers a potential explanation as to why rituximab is clinically highly efficacious in autoantibody-mediated diseases despite no accompanying reduction in serum autoantibody levels.
Introduction
The majority of patients with myasthenia gravis (MG) initially present with ocular symptoms, but it is difficult to predict which cases will remain as ocular MG (OMG) or will progress to ...generalized MG. Herein we evaluated the serologic profile of Japanese OMG and its relationship with clinical features.
Methods
Seventy‐three patients with OMG from five Japanese myasthenia gravis (MG) centers were enrolled. Live cell‐based assays (CBAs) were used to determine the presence of autoantibodies (Abs) to clustered adult (2α, β, δ, ε) and fetal (2α, β, δ, γ) acetylcholine receptor (AChR) isoforms, muscle‐specific receptor tyrosine kinase (MuSK), and lipoprotein receptor–related protein‐4 (LRP4).
Results
Thirty‐four of 73 (46.5%) serum samples were positive for Abs against both the adult‐type and fetal‐type AChR, as expected, but 7 (9.6%) and 2 (2.7%) were positive only for fetal or adult AChR‐Abs, respectively. Four (5.4%) samples were positive for MuSK‐Abs, but two of these also contained antibodies to fetal AChR or LRP4. Twenty‐six (35.6%) samples were seronegative.
Discussion
Abs against fetal‐specific AChR, MuSK, and LRP4 are found in some patients with OMG. Future studies attempting to predict conversion from ocular symptoms to generalized MG may benefit from measurement of these antibodies.
Abstract The role of complement has been demonstrated in experimental models of neuromyelitis optica (NMO), however, only few studies have analysed complement components longitudinally in NMO ...patients. We measured serum or plasma concentrations of anti-C1q antibodies and complement split products C3a and C4a and soluble C5b-9 in patients with NMO, multiple sclerosis and healthy controls. NMO patients had higher levels of C3a and anti-C1q antibodies than healthy controls. C3a levels correlated with disease activity, neurological disability and aquaporin-4 IgG in NMO patients suggesting a role of the alternative pathway of complement in the pathogenesis of NMO and supporting the strategy of therapeutic complement inhibition.
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