Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease caused by the aquaporin (AQP)-4-antibody. Pathological studies on NMO have revealed extensive astrocytic damage, as evidenced by the loss of AQP4 and glial fibrillary acidic protein (GFAP), specifically in perivascular regions with immunoglobulin and complement depositions, although other pathological patterns, such as a loss of AQP4 without astrocyte destruction and clasmatodendrosis, have also been observed. Previous studies have shown that complement-dependent antibody-mediated astrocyte lysis is likely a major pathomechanism in NMO. However, there are also data to suggest antibody-mediated astrocyte dysfunction in the absence of complement. Thus, the importance of complement inhibitory proteins in complement-dependent AQP4-antibody-mediated astrocyte lysis in NMO is unclear. In most of the previous studies, the complement and target cells (astrocytes or AQP4-transfected cells) were derived from different species; however, the complement inhibitory proteins that are expressed on the cell surface cannot protect themselves against complement-dependent cytolysis unless the complements and complement inhibitory proteins are from the same species. To resolve these issues, we studied human astrocytes in primary culture treated with AQP4-antibody in the presence or absence of human complement and examined the effect of complement inhibitory proteins using small interfering RNA (siRNA). METHODS: Purified IgG (10 mg/mL) was obtained from 5 patients with AQP4-antibody-positive NMO, 3 patients with multiple sclerosis (MS), and 3 healthy controls. Confluent human astrocytes transfected with Venus-M1-AQP4-cDNA were incubated with IgG (5% volume). After washing, we cultured the cells with human complements with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes using a fluorescence microscope. We also evaluated cytotoxicity using a propidium iodide (PI) kit and the lactate dehydrogenase (LDH) assay. RESULT: AQP4-antibody alone caused clustering and degradation followed by endocytosis of membraneous AQP4, thereby resulting in decreased cellular adherence and the shrinkage of astrocytic processes. However, these changes were partially reversed by the removal of IgG in culture. In contrast, following the application of AQP4-antibody and non-heated human complements, the cell bodies and nuclei started to swell. At 3 h, most of the astrocytes had lost mobility and adherence and were eventually destroyed or had swollen and were then destroyed. In addition, the remaining adherent cells were mostly PI-positive, indicating necrosis. Astrocyte lysis caused by rabbit complement occurred much faster than did cell lysis with human complement. However, the cell lysis was significantly enhanced by the transfection of astrocytes with siRNA against human CD55 and CD59, which are major complement inhibitory proteins on the astrocyte membrane. AQP4-antibody-negative IgG in MS or control did not induce such changes. CONCLUSION: Taken together, these findings suggest that both complement-dependent and complement-independent AQP4-antibody-mediated astrocytopathies may operate in NMO, potentially contributing to diverse pathological patterns. Our results also suggest that the effect of complement inhibitory proteins should be considered when evaluating AQP4-antibody-mediated cytotoxicity in AQP4-expressing cells.

A randomized, controlled trial of fingolimod (FTY720) in Japanese patients with multiple sclerosis.

BACKGROUND: Fingolimod (FTY720) has previously shown clinical efficacy in phase II/III studies of predominantly Caucasian populations with multiple sclerosis (MS). OBJECTIVES: To report six-month efficacy and safety outcomes in Japanese patients with relapsing MS treated with fingolimod. METHODS: In this double-blind, parallel-group, phase II study, 171 Japanese patients with relapsing MS were randomized to receive once-daily fingolimod 0.5 mg or 1.25 mg, or matching placebo for six months. The primary and secondary endpoints were the percentages of patients free from gadolinium (Gd)-enhanced lesions at months 3 and 6, and relapses over six months, respectively; safety outcomes were also assessed. RESULTS: 147 patients completed the study. Higher proportions of patients were free from Gd-enhanced lesions at months 3 and 6 with fingolimod (0.5 mg: 70%, p = 0.004; 1.25 mg: 86%, p < 0.001) than with placebo (40%). Odds ratios for the proportions of relapse-free patients over six months favoured fingolimod versus placebo but were not significant. Adverse events related to fingolimod included transient bradycardia and atrioventricular block at treatment initiation, and elevated liver enzyme levels. CONCLUSIONS: This study demonstrated the clinical efficacy of fingolimod for the first time in Japanese patients with MS, consistent with the established effects of fingolimod in Caucasian patients.

A case of delayed encephalopathy after carbon monoxide poisoning longitudinally monitored by diffusion tensor imaging.

A woman with DE after CO poisoning was longitudinally evaluated by DTI, performed during the following periods: at the phase of acute CO poisoning, the lucid interval, neurologic deterioration due to DE, and neurologic recovery. The present case revealed the long-term course of DTI parameters after CO poisoning and the usefulness of DTI for quantifying neurologic damage after CO poisoning.

Pain in neuromyelitis optica and its effect on quality of life: a cross-sectional study.

OBJECTIVE: To assess the features of pain and its impact on the health-related quality of life (HRQOL) in neuromyelitis optica (NMO). METHODS: We analyzed 37 patients with NMO or NMO spectrum disorders seen at the Department of Neurology, Tohoku University Hospital, Sendai, Japan, during the period from November 2008 to February 2009. A total of 35 of them were aquaporin-4 antibody-positive. We used Short Form Brief Pain Inventory (BPI) to assess pain and Short Form 36-item (SF-36) health survey to evaluate the HRQOL. Fifty-one patients with multiple sclerosis (MS) were also studied for comparison. RESULTS: Pain in NMO (83.8%) was far more common than in MS (47.1%). The Pain Severity Index score in BPI was significantly higher in NMO than in MS, and patients' daily life assessed by BPI was highly interfered by pain in NMO as compared with MS. Pain involving the trunk and both legs was much more frequent in NMO than in MS. SF-36 scores in NMO were lower than MS, especially in bodily pain. CONCLUSION: Our study showed that pain in NMO is more frequent and severe than in MS and that pain has a grave impact on NMO patients' daily life and HRQOL. Therapy to relieve pain is expected to improve their HRQOL.

Astrocytic damage is far more severe than demyelination in NMO: a clinical CSF biomarker study.

INTRODUCTION: Loss of aquaporin 4 and glial fibrillary acidic protein (GFAP) with necrosis and demyelination is a prominent pathologic feature of neuromyelitis optica (NMO). However, the clinicopathologic significance of astrocytic damage and its relation with demyelination are unknown. OBJECTIVE: To analyze clinical and pathologic values of a CSF biomarker of astrocytic damage in NMO. METHODS: We measured the levels of GFAP, S100B, myelin basic protein (MBP), and neurofilament H (NF-H) in CSF obtained from patients with NMO (n = 33), multiple sclerosis (MS) (n = 27), acute disseminated encephalomyelitis (ADEM), ischemia, meningitis, and other neurologic disease controls (OND). RESULTS: The CSF-GFAP levels during relapse in NMO (2,476.6 +/- 8,815.0 ng/mL) were significantly higher than those in MS (0.8 +/- 0.4 ng/mL) and OND (0.7 +/- 0.5 ng/mL), and much beyond those in ADEM (14.1 +/- 27.4 ng/mL). The sensitivity and specificity of CSF-GFAP for NMO was 90.9% and 76.9% in all, but the specificity improved above 90% in cases limited to demyelinating diseases. CSF-S100B showed a similar trend but was less remarkable. In contrast, MBP and NF-H are not different between NMO and MS. Following treatments, the CSF-GFAP rapidly decreased to a normal level, but CSF-MBP remained high. There were strong correlations between the CSF-GFAP, CSF-S100B, or CSF-MBP levels and Expanded Disability Status Scale (EDSS) or spinal lesion length in the acute phase (r > 0.6). Only CSF-GFAP correlated with EDSS at 6-month follow-up (r = 0.51) in NMO. CONCLUSIONS: Astrocytic damage reflected by elevated CSF glial fibrillary acidic protein is a clinically relevant, primary pathologic process in neuromyelitis optica, and is far more severe than demyelination.

Neuromyelitis optica preceded by hyperCKemia episode.

BACKGROUND: Neuromyelitis optica (NMO) is a disease of the CNS characterized by severe optic neuritis and longitudinally extended transverse myelitis. Recent studies suggest that anti-aquaporin-4 (AQP4) antibodies, NMO-specific biomarkers, are pathogenic and target AQP4-expressing astrocytes in NMO, although an additional event (T-cell response or infection) should occur for anti-AQP4 antibodies and complements to pass through the blood-brain barrier and cause the CNS lesions. AQP4 is the major water channel in the CNS, but it is also expressed in fast-twitch skeletal muscle fibers. However, muscle diseases have not been described in NMO. METHODS: We retrospectively examined the serologic database of 733 cases of NMO with anti-AQP4 antibody at the Department of Neurology, Tohoku University School of Medicine. The serum samples were sent to our laboratory for testing anti-AQP4 antibody from around the country during the period from 2006 to 2009. RESULTS: We found 3 anti-AQP4 antibody-positive female patients (7, 34, and 67 years old) with NMO who had episodes of prominent hyperCKemia (12,520, 19,415, and 59,660 IU/L) with general fatigue some weeks before the onset of optic neuritis. HyperCKemia was transient without any treatment in all patients, but recurred once in one of them. CONCLUSIONS: These cases suggest that hyperCKemia may be involved in the pathogenesis of neuromyelitis optica (NMO) in a fraction of patients. The causes of transient hyperCKemia are unknown. Further studies are needed to know the frequency of hyperCKemia in NMO and clarify its pathogenic role.

Corticolimbic gray matter loss in Parkinson's disease without dementia.

BACKGROUND: The relationship between corticolimbic involvement and cognitive dysfunction in non-demented Parkinson's disease (PD) patients has not yet been elucidated. OBJECTIVES: To delineate involvement of the cerebral cortex and limbic structures in non-demented PD and to clarify distributional differences of gray matter loss between non-demented PD with impaired cognition (PD-CI) and without cognitive impairment (PD-NC). METHODS: Operational criteria based on the Clinical Dementia Rating were used to identify PD-CI. Of 40 consecutive non-demented patients with PD, 13 were classified as PD-CI and 27 as PD-NC. Comparisons of regional gray matter volume (rGMV) were made amongst the PD-CI, PD-NC, and control groups using voxel-based morphometry. RESULTS: Gray matter loss was found extensively in the frontal, temporal, parietal, and occipital cortices in the present non-demented patients with PD. rGMV in the medial frontal and medial occipital cortices was reduced comparably in the PD-NC and PD-CI groups. The severity of gray matter loss in the perisylvian cortices increased in order from the control, to the PD-NC, to the PD-CI groups. rGMV reduction in the lateral and orbital frontal, medial and lateral temporal, medial and lateral parietal, and lateral occipital cortices and cerebellum was found specifically in PD-CI. CONCLUSIONS: Our results suggest that corticolimbic degeneration occurs in non-demented patients with PD, and extensive involvement of the limbic and posterior cortical regions as well as the frontal cortices is associated with cognitive impairment in PD.

CSF protein biomarkers for proximal axonal damage improve prognostic accuracy in the acute phase of Guillain-Barré syndrome.

Early predictors of prognosis in Guillain-Barré syndrome (GBS) are needed to identify patients who are likely to make a poor recovery and to guide therapeutic decision-making in the acute phase. Here we investigate whether axonal protein biomarkers released into the cerebrospinal fluid (CSF) following proximal axonal damage improve the early prognostic accuracy in GBS. A prospective multicenter study including 132 patients (38 GBS, 38 neurological controls, 42 headaches, 14 chronic inflammatory demyelinating neuropathy). CSF levels of axonal [neurofilament (NfH) and tau] and glial (S100B and glial fibrillary acidic protein) protein biomarkers were measured on admission. Nerve conduction studies were performed at the time of lumbar puncture and patients were classified according to neurophysiological criteria. Outcome was assessed on the Hughes functional score (F-score). Poor outcome was defined as the inability to walk independently (F-score > or = 3). High NfH levels (>0.73 ng/ml) predicted poor outcome (P = 0.01) with an odds ratio of 7.3 and correlated with the outcome F-score (R = 0.51, P < 0.01), as did hTau levels (R = 0.47, P < 0.01). Patients with poor outcome had significantly higher CSF NfH (median 1.78 ng/ml) when compared to those with good outcome (0.03 ng/ml) or all of the control groups (neurological controls 0.18 ng/ml, headaches 0.06 ng/ml, chronic inflammatory demyelinating neuropathy 0.05 ng/ml). Except for age (P < 0.05) and need for ventilatory support (P < 0.05), none of the other features reliably predicted outcome. Improved prognostic accuracy in the acute phase of GBS seems possible using CSF NfH levels.

Impact of serotonin transporter gene polymorphism on brain activation by colorectal distention.

BACKGROUND AND AIMS: Determining the gene that plays a key role in brain-gut interactions is a crucial step for clarifying the pathophysiology of irritable bowel syndrome (IBS). We previously reported that the 5-hydroxytryptamine transporter gene-linked polymorphic region (5-HTTLPR) is related to anxiety in subjects with IBS. The amygdala is more activated during fearful face recognition in individuals with the s allele of 5-HTTLPR. Here, we tested our hypothesis that 5-HTTLPR differentially activates brain regions with colorectal distention in humans. METHODS: We enrolled 28 subjects without any organic disease. The study was approved by the Ethics Committee and all subjects gave written informed consent. DNA was extracted from the peripheral blood. The genotype of 5-HTTLPR was determined using polymerase chain reaction. Age, sex, diagnosis-matched individuals with the s/s genotype (n=14) and individuals with the l allele (genotypes l/s, l/l, l/extra-l, n=14) were compared. A barostat bag was inserted to the colorectum and was intermittently inflated with no (0 mm Hg), mild (20 mm Hg), or intense (40 mm Hg) stimulation on a random order. Radioactive H2[(15-)O] saline was injected at bag inflation and then positron emission tomography was performed. Changes in rCBF were analyzed using statistical parametric mapping. RESULTS: Individuals with the s/s genotype showed a significantly larger increase in rCBF by colorectal distention from 0 mm Hg to 40 mm Hg than individuals with the l allele. The significantly more activated brain regions in individuals with the s/s genotype were the left anterior cingulate cortex and right parahippocampal gyrus (p<0.0001). The increase in rCBF by colorectal distention of 20 mm Hg compared with 0 mm Hg was significantly larger in the left orbitofrontal cortex of individuals with the s/s genotype than that of individuals with the l allele (p<0.0001). CONCLUSION: These data suggest that individuals with a weak function of serotonin transporter respond to gut signals more in emotion-regulating brain regions. Functional gene polymorphism may partially predict the individual effect of a selective serotonin reuptake inhibitor on visceral pain.

Occurrence of acute large and edematous callosal lesions in neuromyelitis optica.

BACKGROUND: The corpus callosum is commonly involved in multiple sclerosis (MS), but the characteristics of callosal lesions in neuromyelitis optica (NMO) are unknown.ObjectiveTo reveal the features of callosal lesions in NMO in comparison to MS. METHODS: We retrospectively reviewed the medical records and the brain magnetic resonance imaging films of 56 patients with MS and 22 patients with NMO. RESULTS: In MS, 36 (64.3%) of 56 patients had callosal lesions, but only four patients had acute lesions. All such acute lesions were small, isolated and non-edematous, and the intensity was homotonic. Chronic lesions were observed in 34 patients with MS, and 32 (94%) of them presented small lesions located at the callosal lower margin ("hemi-oval pattern"). Meanwhile, four (18.2%) patients with NMO had callosal lesions, and three of them had acute lesions. Those acute lesions were multiple, large edematous ones with heterogeneous intensity ("marbled pattern"). In the chronic stage, the lesions shrank or disappeared. CONCLUSIONS: Acute large, edematous callosal lesions occasionally occur in NMO. Similar to longitudinally extensive transverse myelitis, such callosal lesions may reflect severe edematous inflammation in NMO, and may provide additional evidence that the pathogenesis in NMO is different from that in MS.

Marked increase in cerebrospinal fluid glial fibrillar acidic protein in neuromyelitis optica: an astrocytic damage marker.

BACKGROUND: Neuromyelitis optica (NMO) is a neurological inflammatory disease associated with autoimmunity to aquaporin 4, predominantly localised in astrocytic foot processes. Recent studies have revealed that loss of aquaporin 4 and glial fibrillar acidic protein (GFAP) is a prominent feature of NMO lesions, suggesting astrocytic impairment. OBJECTIVE: To reveal a useful clinical biomarker of NMO. METHODS: Enzyme-linked immunosorbent assays were carried out for astrocytic markers GFAP and S100B in CSFs, obtained from the patients with NMO (n = 10) and multiple sclerosis (MS) (n = 10) manifesting acute myelitis, acute disseminated encephalomyelitis (ADEM) (n = 3), spinal infarction (n = 3), and other neurological diseases (OND) (n = 5). RESULTS: The CSF-GFAP levels during relapse in NMO (7666.0 (SD 15 266.5) ng/ml) were significantly over several thousand times higher than those in MS (0.7 (1.5)) or OND (0.6 (0.3)), and considerably higher than those in spinal infarction (354.7 (459.0)) and ADEM (0.4 (0.2)). They returned close to normal levels along with clinical improvement soon after corticosteroid therapy in NMO. There were strong correlations between the CSF-GFAP or S100B levels and expanded disability status scales or spinal lesion length in NMO (r>0.9). CONCLUSIONS: CSF-GFAP and S100B may be clinically useful biomarkers in NMO, and astrocytic damage is strongly suggested in the acute phase of NMO.

Temporal changes and geographical differences in multiple sclerosis phenotypes in Japanese: nationwide survey results over 30 years.

BACKGROUND: There are two distinct phenotypes of multiple sclerosis (MS) in Asians, manifesting as optic-spinal (OSMS) and conventional (CMS) forms. In Japan, four nationwide surveys of MS have been conducted. The first three were in 1972, 1982, and 1989, and we performed the fourth in 2004. RESULTS: The recent survey showed six main findings as follows: (1) a four-fold increase in the estimated number of clinically definite patients with MS in 2003 (9900; crude MS prevalence, 7.7/100,000) compared with 1972; (2) a shift in the peak age at onset from early 30s in 1989 to early 20s in 2003; (3) a successive proportional decrease in optic-spinal involvement in clinically definite patients with MS; (4) a significant north-south gradient for the CMS/OSMS ratio; (5) after subdivision of the mainland (30-45 degrees North) into northern and southern parts at 37 degrees N, northern-born northern residents (northern patients) showed a significantly higher CMS/OSMS ratio and higher frequency of brain lesions fulfilling the Barkhof criteria (Barkhof brain lesions) than southern-born southern residents (southern patients); (6) among northern patients, the absolute numbers of patients with CMS and those with Barkhof brain lesions rapidly increased with advancing birth year. CONCLUSIONS: These findings suggest that MS phenotypes are drastically altered by environmental factors, such as latitude and "Westernization."

Intractable hiccup and nausea in neuromyelitis optica with anti-aquaporin-4 antibody: a herald of acute exacerbations.

BACKGROUND: Intractable hiccup and nausea (IHN) are unique symptoms in neuromyelitis optica (NMO). Recent studies have strongly suggested that the pathogenesis of NMO is closely associated with anti-aquaporin-4 (AQP4) antibody. However, clinical implications of IHN and the relationship with anti-AQP4 antibody remain unknown. METHODS: The past medical records of 35 patients with seropositivity for anti-AQP4 antibody were reviewed. We also followed the titres of anti-AQP4 antibody in a patient with NMO, who had newly developed IHN. RESULTS: Of the 35 patients, 15 patients (43%) had episodes of IHN. There was a total of 35 episodes of IHN in these 15 patients and, of the 35 episodes, hiccup was seen in 23 episodes (66%) and nausea was seen in 28 episodes (80%). The IHN frequently preceded (54%) or accompanied (29%) myelitis or optic neuritis. The IHN was often preceded by an episode of viral infection. The titres of anti-AQP4 antibody were remarkably increased when the intractable hiccup appeared in a case. CONCLUSIONS: IHN could be a clinical marker for the early phase of an exacerbation. Careful observation may be needed when INH is seen in patients with NMO, and the early initiation of the treatment could prevent subsequent neurological damage.

Preferential spinal central gray matter involvement in neuromyelitis optica. An MRI study.

OBJECTIVE: To delineate the MRI features that distinguish neuromyelitis optica (NMO) from multiple sclerosis (MS). METHODS: We compared the distribution of the spinal cord lesions by analyzing 1) lesion area, 2) lesion density (by superimposing the lesions onto the standard sections of the cervical and thoracic cord with appropriate transparencies using computer software), and 3) T1-hypointensity in axial sections of MRI in NMO and MS. RESULTS: In NMO, 60-70% of the cervical and thoracic cord MRI lesions occupied more than half of the cord area and mainly involved the central gray matter in the acute stage. In the chronic stage, half or more of the lesions were localized at the central gray matter region. The lesion superimposition analysis also revealed much higher densities in the central gray matter region than in the peripheral white matter regions. Two patients with NMO had T1-hypointense lesions in the central region. In contrast, over 80% of the lesions in MS were localized in the lateral and posterior white matter regions of the cord in the chronic as well as acute stage. Lesion densities were much higher in the lateral and posterior white matter regions than in the central gray matter region. None of the lesions in MS were T1-hypointense. CONCLUSIONS: These MRI findings strongly suggest a preferential involvement in the spinal central gray matter in NMO which is distinct from MS.

Modulation of dendritic cell development by immunoglobulin G in control subjects and multiple sclerosis patients.

Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 alpha4-integrin], the adhesion molecule required for mDCs to cross the blood-brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.

Low-dose corticosteroids reduce relapses in neuromyelitis optica: a retrospective analysis.

Neuromyelitis optica (NMO) is a relapsing neurologic disease characterized by severe optic neuritis and transverse myelitis. A disease-modifying therapy for NMO has not been established. We retrospectively analysed the effect of low-dose corticosteroid (CS) monotherapy on the annual relapse rate in nine patients with NMO. We divided the clinical course in each patient into two periods; the CS Period in which CS was administered, and the No CS Period in which CS was not administered. Periods related to other immunological therapies, such as high-dose methylprednisolone, immunosuppressants, interferon-beta, and plasma exchange, were excluded. As a result, the annual relapse rate during the CS Periods [median, 0.49 (range, 0-1.31)] was found to be significantly lower than that during the No CS Periods [1.48 (0.65-5.54)]. As for the dose of CS, relapses occurred significantly more frequently with ;10 mg/day or less' than with ;over 10 mg/day' (odds ratio: 8.75). The results of the present study suggest a beneficial effect of low-dose CS monotherapy in reducing relapses in NMO.

Tracheostomy can fatally exacerbate sleep-disordered breathing in multiple system atrophy.

Tracheostomy has been employed to release the airway obstruction at the glottic level and to prevent sudden death in patients with multiple system atrophy (MSA). However, sudden death is possible even after tracheostomy. Nocturnal polysomnography showed that the apnea-hypopnea index became higher after tracheostomy, and all tracheostomized patients had frequent central sleep apneas.

Clinical and laboratory features of neuromyelitis optica with oligoclonal IgG bands.

Of 23 neuromyelitis optica (NMO) cases, we found two cases with oligoclonal IgG bands (OBs). Both patients were positive for NMO-IgG. Their common features were long disease duration and co-existing autoimmune diseases (myasthenia gravis and sicca syndrome). Although OBs are mostly negative in NMO, which distinguishes it from multiple sclerosis (MS), they can be positive by long-standing autoimmunity, which may not be directly related to NMO.