anti-AQP4 IgG Research Articles

Peripheral memory B cells in multiple sclerosis vs. double negative B cells in neuromyelitis optica spectrum disorder: disease driving B cell subsets during CNS inflammation.

B cells are fundamental players in the pathophysiology of autoimmune diseases of the central nervous system, such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). A deeper understanding of disease-specific B cell functions has led to the differentiation of both diseases and the development of different treatment strategies. While NMOSD is strongly associated with pathogenic anti-AQP4 IgG antibodies and proinflammatory cytokine pathways, no valid autoantibodies have been identified in MS yet, apart from certain antigen targets that require further evaluation. Although both diseases can be effectively treated with B cell depleting therapies, there are distinct differences in the peripheral B cell subsets that influence CNS inflammation. An increased peripheral blood double negative B cells (DN B cells) and plasmablast populations has been demonstrated in NMOSD, but not consistently in MS patients. Furthermore, DN B cells are also elevated in rheumatic diseases and other autoimmune entities such as myasthenia gravis and Guillain-Barré syndrome, providing indirect evidence for a possible involvement of DN B cells in other autoantibody-mediated diseases. In MS, the peripheral memory B cell pool is affected by many treatments, providing indirect evidence for the involvement of memory B cells in MS pathophysiology. Moreover, it must be considered that an important effector function of B cells in MS may be the presentation of antigens to peripheral immune cells, including T cells, since B cells have been shown to be able to recirculate in the periphery after encountering CNS antigens. In conclusion, there are clear differences in the composition of B cell populations in MS and NMOSD and treatment strategies differ, with the exception of broad B cell depletion. This review provides a detailed overview of the role of different B cell subsets in MS and NMOSD and their implications for treatment options. Specifically targeting DN B cells and plasmablasts in NMOSD as opposed to memory B cells in MS may result in more precise B cell therapies for both diseases.

Quantitative Magnetic Resonance Imaging Analysis of Early Markers of Upper Cervical Cord Atrophy in Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder.

Purpose To quantitatively analyze the C2/C3 segments of the spinal cord on magnetic resonance imaging (MRI) scans of neuromyelitis optica spectrum disorder (NMOSD) and relapsing-remitting multiple sclerosis (RRMS) patients in their first five years of the disease and to investigate the intergroup differences regarding markers of spinal cord atrophy and their correlations with expanded disability status scale (EDSS). Materials and Methods Twenty NMOSD patients and twenty RRMS patients, within their first five years of the disease, were enrolled in this cross-sectional study. All patients underwent spinal cord MR imaging using 1.5 Tesla systems, and C2/C3 portions of the spinal cord were segmented in the obtained scans. C2/C3 anteroposterior diameter (C2/C3 SC-APD), transversal diameter (C2/C3 SC-TD), and cross-sectional area (C2/C3 SC-CSA) were quantitatively measured using Spinal Cord Toolbox v.4.3. Results Three NMOSD patients were seropositive for anti-AQP4 IgG. The mean C2/C3 SC-CSA in NMOSD patients was significantly lower than in RRMS patients. NMOSD patients had significantly lower C2/C3 SC-TDs than RRMS patients. With the three anti-AQP4+ patients excluded from the analysis, C2/C3 SC-TD was negatively correlated with EDSS. Conclusion In the early stages of the disease, quantitative evaluation of C2/C3 spinal cord parameters, including cross-sectional area and transversal diameter in NMOSD patients, appears to be of potential diagnostic and prognostic value.

A prospective, observational study on conversion of clinically isolated syndrome to multiple sclerosis during 4-year period (MS NEO study) in Taiwan.

ImportanceCIS to MS conversion rates vary depending on population cohorts, initial manifestations, and durations of follow-up.ObjectiveTo investigate conversion rate of patients from CIS to MS and the prognostic significance of demographic and clinical variables in Taiwanese population.DesignNationwide, prospective, multi-centric, observational study from November 2008 to November 2014 with 4 years follow-up.SettingMulti-centre setting at 5 institutions in Taiwan.Participants152 patients having single clinical event potentially suggestive of MS in last 2 years were enrolled as consecutive sample. 33 patients were lost to follow-up and 16 patients did not complete the study.103 patients completed the study.Intervention(s) (for clinical trials) or exposure(s) (for observational studies)Natural progression from first episode of CIS to MS or NMO was observed.Main outcome(s) and measure(s)Variables analysed were ‘proportion of patients converting to MS or NMO after first episode of CIS’, ‘duration between first episode of neurological event and diagnosis of MS’, ‘status of anti-AQP4 IgG’ and ‘length of longest contiguous spinal cord lesion in MS patients’. Association between baseline characteristics and progression to MS from CIS was analyzed using multiple logistic regression. Multivariate time dependent effect of baseline characteristics on progression to MS was plotted.Results14.5% patients with CIS converted to MS after 1.1 ± 1.0 years with greater predisposition (18.8%) in those having syndromes referable to the cerebral hemispheres. Conversion rate from ON to MS was 9.7%. 90.9% patients had mild disease course. 46.7% patients had abnormal MRIs at baseline, with 0.6±0.5 contrast enhanced lesions. ‘Below normal BMI’ and ‘MRI lesion load (≥ 4 lesions)’ were identified as risk indicators for the development of MS. Amongst the patients who developed NMO as diagnosed by modern criteria, 80% were positive for anti-AQP4 IgG antibody.Conclusions and relevance‘Below normal BMI’ and ‘number of demyelinating lesions (≥4)’ are significant predictors of conversion from CIS to MS. A low conversion rate to MS in Taiwanese CIS patients and majority of them having a mild course and minimal disability suggest the roles of geographic, genetic and ethnic factors.Trial registrationNon-trial observational study.

Inhibitors of Mammalian Aquaporin Water Channels.

Aquaporins (AQPs) are water channel proteins that are essential to life, being expressed in all kingdoms. In humans, there are 13 AQPs, at least one of which is found in every organ system. The structural biology of the AQP family is well-established and many functions for AQPs have been reported in health and disease. AQP expression is linked to numerous pathologies including tumor metastasis, fluid dysregulation, and traumatic injury. The targeted modulation of AQPs therefore presents an opportunity to develop novel treatments for diverse conditions. Various techniques such as video microscopy, light scattering and fluorescence quenching have been used to test putative AQP inhibitors in both AQP-expressing mammalian cells and heterologous expression systems. The inherent variability within these methods has caused discrepancy and many molecules that are inhibitory in one experimental system (such as tetraethylammonium, acetazolamide, and anti-epileptic drugs) have no activity in others. Some heavy metal ions (that would not be suitable for therapeutic use) and the compound, TGN-020, have been shown to inhibit some AQPs. Clinical trials for neuromyelitis optica treatments using anti-AQP4 IgG are in progress. However, these antibodies have no effect on water transport. More research to standardize high-throughput assays is required to identify AQP modulators for which there is an urgent and unmet clinical need.

Rituximab-induced hypogammaglobulinemia in patients with neuromyelitis optica spectrum disorders

ObjectiveTo evaluate the long-term effects of rituximab (RTX) on total and specific immunoglobulins (Igs) in patients with neuromyelitis optica spectrum disorders (NMOSDs).MethodsTotal IgG, IgA, and IgM levels were evaluated in 15 patients with NMOSDs treated with RTX (median follow-up 70 months). Anti-aquaporin 4 (AQP4)-IgG titration was performed on samples from 9 positive patients. Anti-tetanus (TET), anti-varicella-zoster virus (VZV), and anti-Epstein–Barr virus nuclear antigen (EBNA) IgGs were also tested in patients with NMOSDs and in 6 healthy controls (HCs).ResultsRTX reduced total IgG by 0.42 g/L per year, IgA by 0.08 g/L per year, and IgM by 0.07 g/L per year. Hypogammaglobulinemia (hypo-IgG) (IgG < 7 g/L) developed in 11/15 patients. Severe hypo-IgG (IgG < 4 g/L) was found in 3/15 patients, of whom 2 patients developed serious infectious complications. In group analysis, anti-AQP4 IgG titers were reduced by RTX over time, and a significant correlation between anti-AQP4 IgG titers and total IgG levels was found. The effects of RTX were observed on pathogen-specific IgGs as well. In particular, the levels of anti-TET IgG in patients were significantly lower than those in HCs. The half-life of anti-TET IgG was reduced by about 50% in patients compared with the general population.ConclusionsLong-term RTX treatment is associated with the risk of hypo-Ig and reduction of anti-TET protection in patients with NMOSDs. Results obtained in this study suggest the importance of monitoring total and specific Ig levels before and during treatment with anti-CD20 drugs to prevent hypo-Ig–related complications and to optimize clinical management.

Neuromyelitis optica spectrum disorders: An update.

Recent advances in the understanding of neuromyelitis optica spectrum of disorders (NMOSD) have expanded. Diagnostic criteria have changed over the years. The clinical spectrum of disease manifestations are now understood to include sites outside the spinal cord and optic nerve. A variety of autoimmune diseases may coexist with this disorder. Non neurological manifestations have been recently reported. Novel biomarkers other than aquoporin 4 Immunoglobulin G (anti AQP4-IgG) have been discovered which may have clinical relevance. In particul myelin associated oligoglycoprotein antibody (MOG-Ab) associated NMOSD may be relatively benign. This update describes some of these new findings highlighting the clinical manifestations, biomarkers associated with the disease and magnetic resonance imaging characteristics of brain and spinal cord.

EFFECT OF TYSABRI AND AQP4-IGG ON AN IN-VITRO MODEL OF BBB

<h3>Background</h3> Neuromyelitis Optica (NMO) is an inflammatory, astrocytopathic, demyelinating disease of the CNS mediated by anti AQP4-IgG that targets the AQP4 protein, the dominant water channel on astrocyte foot processes at the blood brain barrier (BBB). Natalizimumab (NTZ, Tysabri) prevents migration of activated T lymphocytes across the BBB and is effective in preventing inflammation in MS. However, for unknown reasons, NTZ worsens NMO, precipitating relapses. We explored the effect of NTZ on a model of BBB. <h3>Methods</h3> Using an in-vitro culture model of BBB (Human Brain Endothelial Cells (HBEC) and Human Astrocytes) tightness of HBEC layer was measured using Trans-Endothelial Electrical Resistance (TEER). <h3>Results</h3> Addition of NTZ to the model caused TEER values to reduce significantly compared to control. Addition of AQP4-IgG and NTZ caused further significant reduction of TEER. Addition of just the AQP4-IgG did not have any effect. <h3>Conclusion</h3> NTZ apparently lowers surrogate measures of BBB integrity in the presence of AQP4-IgG. Whether this in vitro effect translates to easier access of AQP4-IgG to the AQP4 in-vivo or predisposes to attacks is uncertain and is being explored.

Aquaporin-4 antibody neuromyelitis optica following anti-NMDA receptor encephalitis

Encephalitis associated with anti-N-Methyl-D-Aspartate Receptor (NMDAR) antibodies is an autoimmune encephalopathy often associated with teratoma [1]. Neuromyelitis optica (NMO) is an inflammatory demyelinating disorder of the central nervous system (CNS) associated with antibodies against aquaporin 4 (AQP4) [2]. Here, we report the case of a patient who developed limbic encephalitis (LE) followed by NMO, whose serum harbored anti-NMDAR and anti-AQP4 IgG antibodies. A 50-year-old woman presented with subacute, shortterm memory loss, confusion, and behavioral changes. Routine blood tests, an infectious disease screening, an electroencephalogram (EEG) and cerebral spinal fluid (CSF) tests were all negative, whereas a brain MRI showed a T2-weighted, hyperintense medial temporal cortex. A pelvic mass was detected by computed tomography (CT) scan. Two months later, she underwent a hystero-adnexectomy with the removal of an ovarian teratoma. A diagnosis of paraneoplastic LE was made. Tests for classical onconeural antibodies (Hu, Ri, Ma2, CV2/CMRP5, amphiphysin, Yo) proved negative. Her memory deficit started to improve 4 months after surgery, when she received repeated courses of low-dose oral steroids. One month later, she had only residual retrograde amnesia (Fig. 1e). Five months later, she developed drowsiness, cervical itching, and impaired gait. Within 2 weeks, she had developed paraplegia and MRI showed multiple T2weighted hyperintense lesions in the pons, hypothalamus, medulla oblongata, and cervical spine (Fig. 1a–c). A CSF analysis proved negative (normal white cell count and proteins, and absence of IgG intrathecal synthesis). Plasmaexchange and high-dose intravenous steroids were initiated, resulting in a slow improvement of strength. Nine months later, she presented with left-eye optic neuritis. High-dose intravenous steroids were partially effective. Three months later, a relapse occurred with weakness of the right limbs. An MRI disclosed new lesions in the pons and dorsal spine (Fig. 1d). Her serum and CSF were tested for AQP4-IgG-Ab by indirect immunofluorescence on a commercial assay (Euroimmun, Lubeck, Germany) [3], proving positive on serum (titer 1:100). The test was extended to NMDAR-IgG-Ab, also proving positive on serum (1:32) and on CSF. The patient was treated with plasma-exchange and oral steroids, with benefit. Three months later, her CSF analysis revealed a normal white cell count and increased protein content (84 mg/dl); matching oligoclonal bands were detected in the CSF and serum. Human leukocyte typing disclosed the presence of the class I allele B8 and class II DR3-DQ2 (DRB1*03-DQB1*02). AQP4-IgG-Aband NMDAR-IgG-Ab-positivity were both confirmed, but a decrease in serum Ab titer was shown (both Abs serum titer 1:10; NMDAR-Ab CSF titer 1:3, 2); VGCC-, AMPAR-, GABAbR-, LGI1and CASPR2-Abs tested negative. Treatment with azathioprine was started. Eight months later, her conditions were stable. This patient presented with LE followed by NMO 1 year later, and tested positive for AQP4and NMDAR-Abs. To M. Zoccarato M. F. Pelizza B. Giometto L. Zuliani (&) Department of Neurology, Ospedale Ca’Foncello, Azienda Unita Locale Socio-Sanitaria 9 Treviso, Piazza Ospedale 1, 31100 Treviso, Italy e-mail: luigizuliani77@gmail.com

Evaluation of a Multiparametric Immunofluorescence Assay for Standardization of Neuromyelitis Optica Serology

BackgroundNeuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the central nervous system, which predominantly affects the optic nerves and spinal cord. In a majority of cases, NMO is associated with antibodies to aquaporin-4 (AQP4) (termed NMO-IgG).AimsIn this study, we evaluated a new multiparametric indirect immunofluorescence (IIF) assay for NMO serology.MethodsSera from 20 patients with NMO, 41 patients with multiple sclerosis (MS), 30 healthy subjects, and a commercial anti-AQP4 IgG antibody were tested in a commercial composite immunofluorescence assay (“Neurology Mosaic 17”; Euroimmun, Germany), consisting of five different diagnostic substrates (HEK cells transfected with AQP4, non-transfected HEK cells, primate cerebellum, cerebrum, and optic nerve tissue sections).ResultsWe identified AQP4 specific and non-specific fluorescence staining patterns and established positivity criteria. Based on these criteria, this kit yielded a high sensitivity (95%) and specificity (100%) for NMO and had a significant positive and negative likelihood ratio (LR+ = ∞, LR− = 0.05). Moreover, a 100% inter- and intra-laboratory reproducibility was found.ConclusionsThe biochip mosaic assay tested in this study is a powerful tool for NMO serology, fast to perform, highly sensitive and specific for NMO, reproducible, and suitable for inter-laboratory standardization as required for multi-centre clinical trials.

Quantitative assays for anti-aquaporin-4 antibody with subclass analysis in neuromyelitis optica

Background: To clarify the clinical relevance of anti-aquaporin-4 (anti-AQP4) antibody titers and immunoglobulin (IgG) subclass. Methods: Using a bridging enzyme-linked immunosorbent assay (ELISA), a flow cytometric assay (FCMA) and an immunofluorescence assay (IFA) for anti-AQP4 antibodies, sera from 142 patients with multiple sclerosis (MS) as defined by the McDonald criteria (2005), 29 with neuromyelitis optica (NMO) who fulfilled the 1999 criteria, 19 with recurrent and/or longitudinally extensive myelitis (RM/LM), 86 with other non-inflammatory neurological diseases (OND) and 28 healthy controls (HC) were studied. Results: Anti-AQP4 antibody positivity rates by IFA, FCMA, and ELISA were 41.4%, 51.7% and 48.3%, respectively, in NMO (1999) patients, and 0% in the OND and HC groups. Twenty-six MS patients (18.3%) were positive for the antibody; 17 met the 2006 NMO criteria, including positivity for anti-AQP4 antibody, and five had longitudinally extensive myelitis (LM). Among the cases with anti-AQP4 antibody detected by FCMA, IgG1, 2, 3, and 4 anti-AQP4 antibodies were found in 97.8%, 37.0%, 6.5% and 6.5% respectively. There was no association of either antibody positivity or level of anti-AQP4 antibody IgG subclasses with clinical parameters after adjustment of p values for multiple comparisons. Conclusions: FCMA and bridging ELISA are useful for detecting and quantifying anti-AQP4 antibodies.

Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes

Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ~20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm(2) with decreasing M1:M23 ratio from 1:1 to 1:3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs with an area of 2142±829 nm(2), which increased to 5137±1119 nm(2) with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ~10(-12) cm(2)/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.

Successful treatment of a woman with relapsing neuromyelitis optica by interferon beta

Neuromyelitis optica (NMO) is an antibody-mediated disease with the target antigen being the water channel aquaporin-4 (AQP4). More than 90% of NMO patients have relapse. Optimal treatments to prevent recurrence have not been established. Previous reports suggest that interferon beta (IFNB) has not been effective in NMO. However, we present a case of NMO with positive anti-myelin oligodendrocyte glycoprotein (MOG)(27-38) IgG and anti-AQP4 IgG treated with IFNB-1a successfully. The effect of IFNB on NMO and the role of anti-MOG antibodies in NMO deserve further study.

Detection of NMO-IgG or anti-AQP4 antibodies： a specifity challenge

Objective Antibody against aquaporin-4 (AQP4) is a validated specific biomarker for the neuromyelitis optica (NMO) spectrum disorders. However， the specifity of NMO-IgG/anti-AQP4 has not been systemically tested in CNS infectious diseases. This study was undertaken to investigate whether anti-AQP4 antibody is present in patients with tuberculous meningitis (TBM) and to compare different assays for assessing the seroprevalence of anti-AQP4 antibodies in patients with TBM and NMO spectrum disorders. Methods We set up the conventional NMO-IgG assay， based on indirect immunofluorescent (IIF) reactivity with mouse brain cryosections， and an AQP4-transfected cell-based assay (CBA). IIF we characterized further by dual immunostaining， and the antibody titers were also analyzed. Both assays were used in parallel to test seropositivity of masked serum samples from patients with neuromyelitis optica (NMO， 24)， longitudinally extensive transverse myelitis (LETM， 22)， multiple sclerosis (MS， 30)， pulmonary tuberculosis (PTB， 30)， tuberculosis meningitis (TBM， 46) and healthy controls (HC， 20). Results Serum NMO-IgG/anti-AQP4 antibody was not exclusively restricted to patients with NMO spectrum disorders. The antibody was detected in the majority of serum samples from TBM patients， and titers of anti-AQP4 IgG in TBM patients were higher than that in those with confirmed NMO and LTEM. Using the IIF assay， seropositivity rates for NMO-IgG were 76.1%(35/46) for TBM patients， 62.5%(15/24) for NMO patients， and 59.1%(13/22) for LETM. Using cell-based assays we found antibodies to AQP4 in 91.3%(42/46) of TBM patients， 91.7%(22/24) for NMO patients， and 86.4%(19/22) for LETM. PTB， MS， and HC all had low rates of anti-AQP4 IgG seropositivity in these assays. Conclusions The data we present suggests that aquaporin-4 autoimmunity may reflect a CNS mycobacteria-initiated immune response. This findings challenge the classic view that NMO-IgG is a disease-specific antibody， and suggests NMO-IgG/anti-AQP4 may be a marker of destructive demyelination.

Anti-AQP4 IgG antibodies as biomarkers for early diagnosis of neuromyelitis optica spectrum disorders 1

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