Neuromyelitis Optica Patient Research Articles

CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation.

Chitinase-3-like protein 1 (CHI3L1) is primarily secreted by activated astrocytes in the brain and is known as a reliable biomarker for inflammatory central nervous system (CNS) conditions such as neurodegeneration and autoimmune disorders like neuromyelitis optica (NMO). NMO is an astrocyte disease caused by autoantibodies targeting the astroglial protein aquaporin 4 (AQP4) and leads to vision loss, motor deficits, and cognitive decline. In this study examining CHI3L1's biological function in neuroinflammation, we found that CHI3L1 expression correlates with cognitive impairment in our NMO patient cohort. Activated astrocytes secrete CHI3L1 in response to AQP4 autoantibodies, and this inhibits the proliferation and neuronal differentiation of neural stem cells. Mouse models showed decreased hippocampal neurogenesis and impaired learning behaviors, which could be rescued by depleting CHI3L1 in astrocytes. The molecular mechanism involves CHI3L1 engaging the CRTH2 receptor and dampening β-catenin signaling for neurogenesis. Blocking this CHI3L1/CRTH2/β-catenin cascade restores neurogenesis and improves cognitive deficits, suggesting the potential for therapeutic development in neuroinflammatory disorders.

RNA profiles to distinguish multiple sclerosis (MS) and neuromyelitis optica (NMO) in peripheral whole blood (S9.003)

RNA profiles to distinguish multiple sclerosis (MS) and neuromyelitis optica (NMO) in peripheral whole blood (S9.003)

Prevalence and Direct Costs of Patients with Neuromyelitis Optica: Data from Campania Region in Southern Italy

Aim: The study aimed to estimate the prevalence and direct costs of neuromyelitis optica (NMO) patients in Campania, Italy. Materials & methods: We retrospectively evaluated 53 NMO patients (mean age: 50.9 ± 16.5 years; 34% men) from the Campania Region administrative databases identified through disease exemption codes in 2018 and analyzed the incidence of NMO among the Campania region population and the disease-related cost. Results: The prevalence of NMO was 0.91 per 100,000 individuals. The average regional cost per NMO patient was 10,836.2 euros. The highest cost was related to drugs (60.6%), followed by hospitalizations (32.7%), diagnostics (4.8%) and laboratory tests (1.0%). Conclusion: NMO is an extremely rare disease with an annual disease-related cost of 0.005% of public health expenditure.

Hematopoietic Stem Cell Transplantation for Children with Neurological Diseases Improves the Outcomes and Not Leads to Significant Late Effects — Report from Russian Group

Strong evidence of safety, tolerability, efficacy and low rate of late effects presence after hematopoietic stem cell transplantation (HSCT) required for children with severe refractory cases of neurological diseases.We aimed to analyze the effectiveness of both allogenic and autologous hematopoietic stem cell transplantation for all cohorts of patients with neurological diseases based on safety, tolerability and late effects rate.Sixty three patients enrolled to the analysis transplanted 2004 - 2017. Diagnosis: Hurler syndrome (HS) - 40 patients, multiple sclerosis (MS) - 13 patients, metochromatic leukodystrophy (ML) - 4 patients, neuromyelitis optica (NMO) - 3 patients. Median of age 11,8 years old (3 months - 17,4 years old). Most of the patients were girls (n=39, 61,8%). All patients were with confirmed diagnosis by clinical and laboratory testing, imagining. Patients with autoimmune disorders (AD) were treated with different immunosuppressive drugs without effect. MS patients received autologous hematopoietic stem cell transplantation (HSCT) with peripheral blood stem cells (PBSC), other patients - allogenic HSCT both from related and unrelated donors. For allogenic HSCT bone marrow (BM) was used in 32 cases, PBSC - in 13 and cord blood (CB) - in 2 cases (for HS only). In 34 cases HSCT from unrelated donor were done. MS patients received Cyclophosphamide and ATG based chemotherapy before autologous HSCT, NMO and ML patients received Treosulfan, Fludarabine, Rituximab and ATG (if unrelated donor). In case of HS myeloablative conditioning (MAC) regimen was used for 29 patients and reduced intensity conditioning (RIC) - for 11 patients. RIC: Fludarabine, Melphalan, ATG. MAC: Busulfan/Treosulfan, Fludarabine, Thiotepa/Melphalan and ATG with Rituximab (in case of unrelated donors). Patients received CsA/Tacro-based with MMF/MTX GvHD prophylaxis for all allogenic cases. In 3 RIC HSCT for HS immunomagnetic СD3/СD19+ depletion of PBSC was used.All MS patients improved fast. Median follow-up of MS pts. 48,7±2,4 months (10-84 months). Two MS patients relapsed (clinical and MRI). Median of engraftment day - +10. No severe complications registered. All patients after allogenic HSCT engrafted (full donor chimerism on day +30). Median of engraftment day - 21 (11-30 days). Thirty nine patients after allogenic HSCT survived. Reasons of death: infections - 4 pts., TRALI - 1 pt., aGvHD - 1 pt. TRM improved, over the years, with improving of supportive care and donor selection as well as pre-transplant screening. No early severe toxicity revealed. GvHD: grade II - 17 patients, grade III-IV - 3 patients, local cGVHD - 3 patients. No patients had extensive cGvHD. All 5 rejected patients were with HS (MAC and RIC rejection rate was same). At median follow up of 60 months (8-160 months), the estimated probability of 5 years OS is 87%. All patients with complete donor's chimerism have good neurological response (best response correlated with early HSCT for all patients). Better status before HSCT correlated with better outcome. Cardio-vascular effects (EF decreasing) were most common for MS patients (n=7), progressive skeletal deformation - for HS patients (totally 17 cases), in one of them operative reconstitution was performed, endocrine (thyroid dysfunction, growth hormone deficiency) - for MS, NMO and HS patients (n=8), late immune reconstitution - for 1 case (NMO patient). Median period of late effects arising after HSCT was 21 month (12-27 months). All survived patients (MS, HS, ML, NMO) improved deficit in neurocognitive sphere in different grade but needed in special rehabilitation.Thus, this is the first analysis of outcomes and late effects of a group of children received HSCT for neurological diseases, especially for AD. HSCT is successful approach for patients with such diseases. In-time HSCT can significantly improve the outcome. Most of the patients remain in remission during the long time of follow-up. Long-time follow-up showed that these patients require the special observational protocol including estimation of cardio-vascular, skeletal, endocrine and neurocognitive risks. Better neurocognitive response correlated with intensive rehabilitation using computer model. Russian Joint study showed effective cooperation for treatment of such patients in the national setting. DisclosuresNo relevant conflicts of interest to declare.

Astrocyte-microglia interaction drives evolving neuromyelitis optica lesion.

Neuromyelitis optica (NMO) is a severe inflammatory autoimmune CNS disorder triggered by binding of an IgG autoantibody to the aquaporin 4 (AQP4) water channel on astrocytes. Activation of cytolytic complement has been implicated as the major effector of tissue destruction that secondarily involves myelin. We investigated early precytolytic events in the evolving pathophysiology of NMO in mice by continuously infusing IgG (NMO patient serum-derived or AQP4-specific mouse monoclonal), without exogenous complement, into the spinal subarachnoid space. Motor impairment and sublytic NMO-compatible immunopathology were IgG dose dependent, AQP4 dependent, and, unexpectedly, microglia dependent. In vivo spinal cord imaging revealed a striking physical interaction between microglia and astrocytes that required signaling from astrocytes by the C3a fragment of their upregulated complement C3 protein. Astrocytes remained viable but lost AQP4. Previously unappreciated crosstalk between astrocytes and microglia involving early-activated CNS-intrinsic complement components and microglial C3a receptor signaling appears to be a critical driver of the precytolytic phase in the evolving NMO lesion, including initial motor impairment. Our results indicate that microglia merit consideration as a potential target for NMO therapeutic intervention.

Membrane assembly of aquaporin-4 autoantibodies regulates classical complement activation in neuromyelitis optica.

Neuromyelitis optica (NMO) is an autoimmune CNS disorder mediated by pathogenic aquaporin-4 (AQP4) water channel autoantibodies (AQP4-IgG). Although AQP4-IgG-driven complement-dependent cytotoxicity (CDC) is critical for the formation of NMO lesions, the molecular mechanisms governing optimal classical pathway activation are unknown. We investigated the molecular determinants driving CDC in NMO using recombinant AQP4-specific autoantibodies (AQP4 rAbs) derived from affected patients. We identified a group of AQP4 rAbs targeting a distinct extracellular loop C epitope that demonstrated enhanced CDC on target cells. Targeted mutations of AQP4 rAb Fc domains that enhance or diminish C1q binding or antibody Fc-Fc interactions showed that optimal CDC was driven by the assembly of multimeric rAb platforms that increase multivalent C1q binding and facilitate C1q activation. A peptide that blocks antibody Fc-Fc interaction inhibited CDC induced by AQP4 rAbs and polyclonal NMO patient sera. Super-resolution microscopy revealed that AQP4 rAbs with enhanced CDC preferentially formed organized clusters on supramolecular AQP4 orthogonal arrays, linking epitope-dependent multimeric assembly with enhanced C1q binding and activation. The resulting model of AQP4-IgG CDC provides a framework for understanding classical complement activation in human autoantibody-mediated disorders and identifies a potential new therapeutic avenue for treating NMO.

Complications and Toxicities Associated with Autologous Stem Cell Transplantation for Severe Autoimmune Diseases: Single Center Experience

BACKGROUNDIn the past 20 years, autologous hematopoietic stem cell transplantation (ASCT) has opened new avenues in the treatment of patients with severe, treatment-resistant autoimmune diseases with the aim of resetting the patient's immune system. Over the years, this has led to increased safety and efficacy of the procedure however it is still associated with morbidity and mortality. We evaluated the complications and toxicities associated with ASCT for autoimmune diseases at The Ottawa Hospital.METHODSUsing the Ottawa stem cell transplant database, consecutive patients undergoing ASCT for autoimmune diseases were identified between 1999 and 2017. Through retrospective chart review, infectious complications, toxicities, length of hospital stay and survival outcomes were extracted.RESULTSNinety patients underwent ASCT for an autoimmune disease at The Ottawa Hospital between 1999 and 2017 (median age 35 years, range 19-65 and 61% female). Of the 90 patients transplanted, 75 (83.3%) had a neurologic disease (53 with multiple sclerosis, 8 with myasthenia gravis, 5 with chronic inflammatory demyelinating polyneuropathy, 4 with neuromyelitis optica, and 5 with stiff person syndrome), 10 (11.1%) had a rheumatological disease (7 scleroderma and 3 rheumatoid arthritis), 4 (4.4%) ad Crohn's disease and 1 (1.1%) had immune thrombocytopenia. The median follow-up time in this study was 3.2 years from date of ASCT (range 0-15.75 years). The median length of stay in hospital was 30 days (range 12-215). The median engraftment time was 10 days for neutrophil recovery and 15 days for platelet recovery.The first 100 days following ASCT included 97 episodes of bacterial infections (28-bacteremia, 8-pneumonia, 29-UTI, 9-C. difficile colitis, 5-neutropenic enterocolitis, 12-skin infections, 4-line infections and 2 sinus/pharyngeal infections), 39 viral reactivations or infections (10-CMV, 7-BK, 2-adenovirus, 2-polyomavirus, 6-EBV, 4-HSV, 4-VZV and 4-respiratory viruses), and 7 episodes of fungal infection (4-mucocutaneous candidiasis, 1-candidemia, 1-yeast on urine culture and 1-C. lusitaneae from sputum).There were 9 patients (10%) that developed acute kidney injury, of which 3 recovered without hemodialysis, 1 recovered with temporary hemodialysis, and 2 required permanent hemodialysis. Ten patients (11.1%) developed liver dysfunction, all of which resolved. There were 17 episodes of hemorrhagic cystitis (5-BK, 2-adenovirus, 2-polyoma virus, 1-CMV, 3 tested negative for viruses, and 4 were untested).The 100-day transplant-related mortality was 2.3% and the overall survival plateaued at 93.5% beyond 2 years (95% CI at 2 years 84.9-97.2%). Two patients developed a secondary malignancy (metastatic adenocarcinoma in a scleroderma patient and acute leukemia in a multiple sclerosis patient that had received mitoxantrone). Five patients have died; 4 from organ toxicity (pulmonary hemorrhage at day -3 in a scleroderma patient, veno-occlusive disease at day 62 in a multiple sclerosis patient, heart failure at day 116 in a scleroderma patient, and bronchiolitis obliterans at day 415 in a neuromyelitis optica patient) and 1 from metastatic adenocarcinoma and advanced scleroderma.CONCLUSIONThis study confirms that the morbidity and mortality of ASCT for patients with autoimmune disease is similar to that for other patient groups undergoing ASCT. Patient selection should take this into consideration. DisclosuresNo relevant conflicts of interest to declare.

Mutations of Recombinant Aquaporin-4 Antibody in the Fc Domain Can Impair Complement-Dependent Cellular Cytotoxicity and Transplacental Transport.

Maternal antibodies provide protection for the developing fetus. Transplacental transport of pathogenic autoantibodies might pose a risk for the developing fetus. The transport of antibodies across the placenta to the fetal circulation occurs through the neonatal Fc salvage receptor (FcRn). During gestation, maternal autoantibodies are able to penetrate the embryonic brain before a functional intact blood–brain barrier is established. Brain-reactive antibodies to the water channel protein aquaporin-4 (AQP4) are a hallmark finding in neuromyelitis optica (NMO), a neurological disease that predominantly affects women, many of whom are of childbearing age. AQP4–IgG mediate astrocytic injury in a complement-dependent fashion. Recent studies suggest these antibodies contribute to impaired pregnancy outcome. The aim of the study was to investigate the transplacental transport as well as FcRn binding of a monoclonal AQP4–IgG cloned from an NMO patient (wild-type antibody) compared to five different mutated Fc domain of this antibody containing single amino acid substitutions in the Fc region. All of the Fc-mutated antibodies lack complement-dependent cytotoxicity. Four of the five Fc-mutated antibodies showed limited transplacental transport in vivo. Three mutated Fc with impaired transplacental transport showed persistent binding to rodent FcRn at pH 6 but also at pH 7.2, suggesting that limited transplacental transport could be due to diminished release from FcRn. One mutated Fc with modestly limited transplacental transport showed diminished binding to FcRn at pH 6. This study suggests that mutated Fc with intact transplacental transport may be used to study antibody effector functions and Fc with limited transport may be used as a carrier to deliver therapies to pregnant woman, while sparing the developing fetus.

Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination

Intrathecal immunoglobulin G (IgG) synthesis, cerebrospinal fluid (CSF) oligoclonal IgG bands and lesional IgG deposition are seminal features of multiple sclerosis (MS) disease pathology. Both the specific targets and pathogenic effects of MS antibodies remain poorly characterized. We produced IgG1 monoclonal recombinant antibodies (rAbs) from clonally-expanded plasmablasts recovered from MS patient CSF. Among these were a subset of myelin-specific MS rAbs. We examined their immunoreactivity to mouse organotypic cerebellar slices by live binding and evaluated tissue injury in the presence and absence of human complement. Demyelination, glial and neuronal viability, and complement pathway activation were assayed by immunofluorescence microscopy and compared to the effects of an aquaporin-4 water channel (AQP4)-specific rAb derived from a neuromyelitis optica (NMO) patient. MS myelin-specific rAbs bound to discrete surface domains on oligodendrocyte processes and myelinating axons. Myelin-specific MS rAbs initiated complement-dependent cytotoxicity to oligodendrocytes and induced rapid demyelination. Demyelination was accompanied by increased microglia activation; however, the morphology and survival of astrocytes, oligodendrocyte progenitors and neurons remained unaffected. In contrast, NMO AQP4-specific rAb initiated complement-dependent astrocyte damage, followed by sequential loss of oligodendrocytes, demyelination, microglia activation and neuronal death. Myelin-specific MS antibodies cause oligodendrocyte loss and demyelination in organotypic cerebellar slices, which are distinct from AQP4-targeted pathology, and display seminal features of active MS lesions. Myelin-specific antibodies may play an active role in MS lesion formation through complement-dependent mechanisms.

Neuromyelitis optica patient relapsed after 9 years with seroreversion of anti‐aquaporin 4 antibodies

Neuromyelitis optica patient relapsed after 9 years with seroreversion of anti‐aquaporin 4 antibodies

Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid.

BackgroundDevic’s neuromyelitis optica (NMO) is an autoimmune astrocytopathy, associated with central nervous system inflammation, demyelination, and neuronal injury. Several studies confirmed that autoantibodies directed against aquaporin-4 (AQP4-IgG) are relevant in the pathogenesis of NMO, mainly through complement-dependent toxicity leading to astrocyte death. However, the effect of the autoantibody per se and the exact role of intrathecal AQP4-IgG are still controversial.MethodsTo explore the intrinsic effect of intrathecal AQP4-IgG, independent from additional inflammatory effector mechanisms, and to evaluate its clinical impact, we developed a new animal model, based on a prolonged infusion of purified immunoglobulins from NMO patient (IgGAQP4+, NMO-rat) and healthy individual as control (Control-rat) in the cerebrospinal fluid (CSF) of live rats.ResultsWe showed that CSF infusion of purified immunoglobulins led to diffusion in the brain, spinal cord, and optic nerves, the targeted structures in NMO. This was associated with astrocyte alteration in NMO-rats characterized by loss of aquaporin-4 expression in the spinal cord and the optic nerves compared to the Control-rats (p = 0.001 and p = 0.02, respectively).In addition, glutamate uptake tested on vigil rats was dramatically reduced in NMO-rats (p = 0.001) suggesting that astrocytopathy occurred in response to AQP4-IgG diffusion. In parallel, myelin was altered, as shown by the decrease of myelin basic protein staining by up to 46 and 22 % in the gray and white matter of the NMO-rats spinal cord, respectively (p = 0.03). Loss of neurofilament positive axons in NMO-rats (p = 0.003) revealed alteration of axonal integrity. Then, we investigated the clinical consequences of such alterations on the motor behavior of the NMO-rats. In a rotarod test, NMO-rats performance was lower compared to the controls (p = 0.0182). AQP4 expression, and myelin and axonal integrity were preserved in AQP4-IgG-depleted condition. We did not find a major immune cell infiltration and microglial activation nor complement deposition in the central nervous system, in our model.ConclusionsWe establish a link between motor-deficit, NMO-like lesions and astrocytopathy mediated by intrathecal AQP4-IgG. Our study validates the concept of the intrinsic effect of autoantibody against surface antigens and offers a model for testing antibody and astrocyte-targeted therapies in NMO.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0577-8) contains supplementary material, which is available to authorized users.

Isotetrandrine Reduces Astrocyte Cytotoxicity in Neuromyelitis Optica by Blocking the Binding of NMO-IgG to Aquaporin 4

Objective: Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease that affects the optic nerves and spinal cord with no cure and no FDA-approved therapy. Research over the last decade revealed that the binding of NMO-IgG to the water channel protein astrocyte aquaporin 4 (AQP4) might be the primary cause of NMO pathogenesis. The purpose of this study was to identify potential blockers of NMO-IgG and AQP4 binding. Methods: We developed a two-step screening platform consisting of a reporter cell-based high-throughput screen assay and a cell viability-based assay. Purified NMO-IgG from NMO patient serum and transfected Chinese hamster lung fibroblast V79 cells stably expressing human M23-AQP4 were used for primary screening of 40,000 small molecule fractions from 500 traditional Chinese herbs. Results: Thirty-six positive fractions were identified, of which 3 active fractions (at 50 μg/ml) were found to be from the same Chinese traditional herb Mahonia japonica (Thunb.). A bioactivity-guided method based on a primary screening assay for blocking activity led to the isolation of an active single natural compound, isotetrandrine, from the 3 fractions. Our immunofluorescence staining results showed that isotetrandrine can block NMO-IgG binding to AQP4 without affecting the expression and function of AQP4. It can also inhibit NMO-IgG binding to astrocyte AQP4 in NMO patient sera and block NMO-IgG-dependent complement-mediated cytotoxicity with the IC₅₀ at ∼3 μ<smlcap>M</smlcap>. Conclusions: The present study developed a cell-based high-throughput screen to identify small molecule inhibitors for NMO-IgG and AQP4 binding, and suggests a potential therapeutic value of isotetrandrine in NMO.

Severely exacerbated neuromyelitis optica rat model with extensive astrocytopathy by high affinity anti-aquaporin-4 monoclonal antibody

IntroductionNeuromyelitis optica (NMO), an autoimmune astrocytopathic disease associated with anti-aquaporin-4 (AQP4) antibody, is characterized by extensive necrotic lesions preferentially involving the optic nerves and spinal cord. However, previous in-vivo experimental models injecting human anti-AQP4 antibodies only resulted in mild spinal cord lesions compared to NMO autopsied cases. Here, we investigated whether the formation of severe NMO-like lesions occurs in Lewis rats in the context of experimental autoimmune encephalomyelitis (EAE), intraperitoneally injecting incremental doses of purified human immunoglobulin-G from a NMO patient (hIgGNMO) or a high affinity anti-AQP4 monoclonal antibody (E5415A), recognizing extracellular domain of AQP4 made by baculovirus display method.ResultsNMO-like lesions were observed in the spinal cord, brainstem, and optic chiasm of EAE-rats with injection of pathogenic IgG (hIgGNMO and E5415A), but not in control EAE. Only in higher dose E5415A rats, there were acute and significantly severer clinical exacerbations (tetraparesis or moribund) compared with controls, within half day after the injection of pathogenic IgG. Loss of AQP4 was observed both in EAE rats receiving hIgGNMO and E5415A in a dose dependent manner, but the ratio of AQP4 loss in spinal sections became significantly larger in those receiving high dose E5415A up to about 50 % than those receiving low-dose E5415A or hIgGNMO less than 3 %. These lesions were also characterized by extensive loss of glial fibrillary acidic protein but relatively preserved myelin sheaths with perivascular deposition of IgG and C5b-9, which is compatible with post mortem NMO pathology. In high dose E5415A rats, massive neutrophil infiltration was observed especially at the lesion edge, and such lesions were highly vacuolated with partial demyelination and axonal damage. In contrast, such changes were absent in EAE rats receiving low-dose E5415A and hIgGNMO.ConclusionsIn the present study, we established a severe experimental NMO rat model with highly clinical exacerbation and extensive tissue destructive lesions typically observed in NMO patients, which has not adequately been realized in in-vivo rodent models. Our data suggest that the pathogenic antibodies could induce immune mediated astrocytopathy with mobilized neutrophils, resulted in early lesion expansion of NMO lesion with vacuolation and other tissue damages. (350/350)Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0259-2) contains supplementary material, which is available to authorized users.

A Distinct Class of Antibodies May Be an Indicator of Gray Matter Autoimmunity in Early and Established Relapsing Remitting Multiple Sclerosis Patients.

*These authors contributed equally to the work in this manuscript.We have previously identified a distinct class of antibodies expressed by B cells in the cerebrospinal fluid (CSF) of early and established relapsing remitting multiple sclerosis (RRMS) patients that is not observed in healthy donors. These antibodies contain a unique pattern of mutations in six codons along VH4 antibody genes that we termed the antibody gene signature (AGS). In fact, patients who have such B cells in their CSF are identified as either having RRMS or developing RRMS in the future. As mutations in antibody genes increase antibody affinity for particular antigens, the goal for this study was to investigate whether AGS+ antibodies bind to brain tissue antigens. Single B cells were isolated from the CSF of 10 patients with early or established RRMS. We chose 32 of these B cells that expressed antibodies enriched for the AGS for further study. We generated monoclonal full-length recombinant human antibodies (rhAbs) and used both immunological assays and immunohistochemistry to investigate the capacity of these AGS+ rhAbs to bind brain tissue antigens. AGS+ rhAbs did not recognize myelin tracts in the corpus callosum. Instead, AGS+ rhAbs recognized neuronal nuclei and/or astrocytes, which are prevalent in the cortical gray matter. This pattern was unique to the AGS+ antibodies from early and established RRMS patients, as AGS+ antibodies from an early neuromyelitis optica patient did not display the same reactivity. Prevalence of CSF-derived B cells expressing AGS+ antibodies that bind to these cell types may be an indicator of gray matter-directed autoimmunity in early and established RRMS patients.

Aquaporin-4 antibody isoform binding specificities do not explain clinical variations in NMO.

Objective:To assess the clinical relevance of the differential binding of antibodies against the 2 main aquaporin-4 (AQP4) isoforms in neuromyelitis optica (NMO) patient sera using stably transfected human embryonic kidney cells.Methods:Flow cytometry of human embryonic kidney cells stably transfected with either M23 or M1 AQP4 was used to measure antibody endpoint titers in 52 remission samples and 26 relapse samples from 34 patients with clinically well-characterized AQP4 antibody–positive NMO/NMO spectrum disorder.Results:The AQP4 M23 (40–61,440) and AQP4 M1 (<20–20,480) titers varied widely between patients, as did the M23:M1 antibody ratio (1–192). In 76 of 78 samples, binding to M23 was higher than binding to M1, including during relapses and remissions (p < 0.0001), and the M23:M1 ratio was relatively constant within an individual patient. Titers usually fell after immunosuppression, but the titers at which relapses occurred varied markedly; no threshold level for relapses could be identified, and relapses could occur without a rise in titers. Relapse severity did not correlate with M23 or M1 antibody titers, although there was a correlation between the earliest M23 titers and annualized relapse rates. The M23:M1 ratio and absolute M23 and M1 titers did not relate to age at disease onset, ethnicity, disease severity, phenotype, or relapses at different anatomical sites.Conclusion:Relative AQP4 antibody binding to M23 and M1 isoforms differs between patients but there is no consistent association between these differences and clinical characteristics of disease. Nevertheless, the M23 isoform provided a slightly more sensitive substrate for AQP4-antibody assays, particularly for follow-up studies.

Cerebrospinal fluid aquaporin-4-immunoglobulin G disrupts blood brain barrier.

To clarify the significance of immunoglobulin G autoantibody specific for the astrocyte water channel aquaporin-4 in cerebrospinal fluid, aquaporin-4-immunoglobulin G from a neuromyelitis optica patient was administered intrathecally to naïve mice, and the distribution and pathogenic impact was evaluated. A distinct distribution pattern of aquaporin-4-immunoglobulin G deposition was observed in the subarachnoid and subpial spaces where vessels penetrate the brain parenchyma, via a paravascular route with intraparenchymal perivascular deposition. Perivascular astrocyte-destructive lesions were associated with blood-borne horseradish peroxidase leakage indicating blood-brain barrier breakdown. The cerebrospinal fluid aquaporin-4-immunoglobulin G therefore distributes widely in brain to initiate astrocytopathy and blood-brain barrier breakdown.

The cerebrospinal fluid immunoglobulin transcriptome and proteome in neuromyelitis optica reveals central nervous system-specific B cell populations.

BackgroundNeuromyelitis optica (NMO) is a severe demyelinating disorder of the central nervous system (CNS) associated with the presence of an autoimmune antibody response (AQP4-IgG) against the water channel aquaporin-4 (AQP4). It remains unclear whether pathologic AQP4-IgG in the CNS is produced entirely by peripheral plasma cells or is generated in part by infiltrating B cells. To determine the overlap of AQP4-IgG idiotypes between the CNS and periphery, we compared the immunoglobulin G (IgG) transcriptome of cerebrospinal fluid (CSF) plasmablasts with the CSF and serum IgG proteomes in 7 AQP4-seropositive NMO patients following exacerbation.MethodsCSF variable region Ig heavy- (VH) and light-chain (VL) transcriptome libraries were generated for each patient from CSF plasmablasts by single cell sorting, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA sequencing. Recombinant antibodies were generated from clonally expanded, paired VH and VL sequences and tested for AQP4-reactivity by cell-binding assay. CSF and serum IgG fractions were searched for sequences that matched their respective CSF IgG transcriptome. Matching peptides within the same patient’s CSF and serum IgG proteomes were also identified.ResultsIn each NMO patient, we recovered CSF IgG VH and VL sequences that matched germline-mutated IgG protein sequences from the patient’s CSF and serum IgG proteomes. Although a modest variation was observed between patients, the overlap between the transcriptome and proteome sequences was found primarily, but not exclusively, within the CSF. More than 50% of the CSF IgG transcriptome sequences were exclusively found in the CSF IgG proteome, whereas 28% were found in both the CSF and blood IgG proteome, and 18% were found exclusively in the blood proteome. A comparable distribution was noted when only AQP4-specific IgG clones were considered. Similarly, on average, only 50% of the CSF IgG proteome matched corresponding peptide sequences in the serum.ConclusionsDuring NMO exacerbations, a substantial fraction of the intrathecal Ig proteome is generated by an intrathecal B cell population composed of both novel and peripherally-derived clones. Intrathecal CSF B cell clones may contribute to NMO disease exacerbation and lesion formation and may be an important target for preventative therapies.

Plasma Homocysteine Levels in Neuromyelitis Optica

Background and purpose: Homocysteine has been implicated in many kinds of neurologic diseases by inducing oxidative injury which is considered one of the pathogenic mechanisms of neuromyelitis optica (NMO). The aim of this study was to investigate whether there were any relationship between plasma homocysteine and clinical features of NMO patients. Methods: We measured plasma homocysteine in 66 patients with NMO and 66 controls. Results: The patients with NMO had significantly higher homocysteine level than that of healthy control (P<0.001). The average homocysteine level of patients with NMO was in normal range. No correlation was found between clinical or magnetic resonance imaging features of patients with NMO and the level of homocysteine. The inactive NMO patients with relapse in followup had significantly higher homocysteine level than those without relapse in follow-up (P=0.045), and the entire inactive NMO patient with relapse had at least one relapse within the first year of followup. Conclusions: The elevated homocysteine levels in NMO patients might be just a secondary change.

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.

Neuromyelitis optica IgG stimulates an immunological response in rat astrocyte cultures

Neuromyelitis optica (NMO) is a primary astrocyte disease associated with central nervous system inflammation, demyelination, and tissue injury. Brain lesions are frequently observed in regions enriched in expression of the aquaporin-4 (AQP4) water channel, an antigenic target of the NMO IgG serologic marker. Based on observations of disease reversibility and careful characterization of NMO lesion development, we propose that the NMO IgG may induce a dynamic immunological response in astrocytes. Using primary rat astrocyte-enriched cultures and treatment with NMO patient-derived serum or purified IgG, we observed a robust pattern of gene expression changes consistent with the induction of a reactive and inflammatory phenotype in astrocytes. The reactive astrocyte factor lipocalin-2 and a broad spectrum of chemokines, cytokines, and stress response factors were induced by either NMO patient serum or purified IgG. Treatment with IgG from healthy controls had no effect. The effect is disease-specific, as serum from patients with relapsing-remitting multiple sclerosis, Sjögren's, or systemic lupus erythematosus did not induce a response in the cultures. We hypothesize that binding of the NMO IgG to AQP4 induces a cellular response that results in transcriptional and translational events within the astrocyte that are consistent with a reactive and inflammatory phenotype. Strategies aimed at reducing the inflammatory response of astrocytes may short circuit an amplification loop associated with NMO lesion development.