Katsuhisa Masaki

Distinct genetic and infectious profiles in Japanese neuromyelitis optica patients according to anti-aquaporin 4 antibody status

Objective To clarify whether genetic and common infectious backgrounds are distinct, according to anti-aquaporin 4 (AQP4) antibody status in Japanese patients with neuromyelitis optica (NMO). Methods We analysed human leucocyte antigen (HLA)-DRB1 and HLA-DPB1 alleles, and IgG antibodies against Helicobacter pylori, Chlamydia pneumoniae, varicella zoster virus and Epstein–Barr virus nuclear antigen (EBNA) in 116 patients with NMO, including 39 patients with neuromyelitis optica spectrum disorder (NMOSD), 145 multiple sclerosis (MS) patients and 367 unrelated healthy controls. 77 NMO/NMOSD patients were seropositive for AQP4 antibody while 39 were seronegative. Results Compared with healthy controls, NMO/NMOSD patients showed a significantly lower frequency of DRB1*0901 and significantly higher frequencies of DRB1*1602 and DPB1*0501, which conferred susceptibility to anti-AQP4 antibody positive NMO/NMOSD, but not antibody negative NMO/NMOSD. DRB1*0901 was a common protective allele, irrespective of the presence or absence of anti-AQP4 antibody. Anti-H pylori and anti-C pneumoniae antibodies were more commonly detected in anti-AQP4 antibody positive NMO/NMOSD patients than healthy controls. Antibody negative NMO/NMOSD patients did not differ from healthy controls regarding the presence of these antibodies. The presence or absence of antibodies against varicella zoster virus and EBNA did not vary among the groups. The frequencies of antibodies against these four pathogens were not significantly different between MS patients and healthy controls. Conclusions Our results suggest that HLA-DRB1*1602 and DPB1*0501 alleles and H pylori and Chlamydia pneumonia infection are risk factors only for anti-AQP4 antibody positive NMO/NMOSD but not for anti-AQP4 antibody negative NMO/NMOSD.

Association of anti-Helicobacter pylori neutrophil-activating protein antibody response with anti-aquaporin-4 autoimmunity in Japanese patients with multiple sclerosis and neuromyelitis optica

There are two distinct subtypes of multiple sclerosis (MS) in Asians: opticospinal (OSMS) and conventional (CMS). OSMS has similar features to neuromyelitis optica (NMO) and half of OSMS patients have the NMO-Immunoglobulin G (IgG)/ anti-aquaporin-4 (AQP4) antibody. We reported that Helicobacter pylori (H. pylori) infection was significantly less common in CMS patients than controls. To reveal the immune responses to the H. pylori neutrophil-activating protein (HP-NAP) in Japanese MS patients, according to anti-AQP4 antibody status, sera from 162 MS patients, 37 patients with other inflammatory neurological diseases (OIND), and 85 healthy subjects were assayed for anti-H. pylori antibodies, anti-HP-NAP antibodies, and myeloperoxidase (MPO) by enzyme immunoassays. H. pylori seropositivity rates were significantly higher in anti-AQP4 antibody-positive MS/NMO (AQP4 + /MS) patients (19/27, 70.4%) than anti-AQP4 antibody-negative CMS (AQP4 — /CMS) patients (22/83, 26.5%). Among H. pylori-infected individuals, the anti-HP-NAP antibody was significantly more common in AQP4 + /MS and AQP4 — /OSMS patients than healthy subjects (36.8%, 34.6% versus 2.8%). Among the AQP4 + /MS patients, a significant positive correlation between anti-HP-NAP antibody levels and the final Kurtzke’s Expanded Disability Status Scale scores was found, and MPO levels were higher in anti-HP-NAP antibody-positive patients than anti-HP-NAP antibody-negative ones. Therefore, HP-NAP may be associated with the pathology of anti-AQP4 antibody-related neural damage in MS/NMO patients.

Reappraisal of brain MRI features in patients with multiple sclerosis and neuromyelitis optica according to anti-aquaporin-4 antibody status

Brain lesions are not uncommon in neuromyelitis optica (NMO) patients with anti-aquaporin-4 (AQP4) antibody; however, the appearance of these lesions is said to be different from that of those in Western patients with multiple sclerosis (MS). To clarify the similarities and dissimilarities of brain lesions in anti-AQP4 antibody-positive and -negative MS and NMO patients, we examined the presence of anti-AQP4 antibody in the sera of 148 consecutive patients fulfilling Posers criteria for clinically definite MS, of whom 38 also met the revised NMO criteria, using an immunofluorescence method, and analyzed brain lesions by magnetic resonance imaging (MRI). Brain lesions fulfilling the Barkhof criteria were significantly more common in 121 patients without anti-AQP4 antibody than in 27 patients with anti-AQP4 antibody (57.0% vs. 33.3%, P=0.033), while the frequency of those that met the Paty criteria was not different between the two groups (74.4% vs. 73.5%). Ovoid lesions were detected more commonly in patients without anti-AQP4 antibody than in those with the antibody (72.3% vs. 48.2%, P=0.022). The anti-AQP4 antibody-positive patients had significantly more atypical brain lesions, such as extensive brain lesions, than the anti-AQP4 antibody-negative ones (18.5% vs. 1.7%, P=0.0023). Thus, although MS-like brain lesions are more common in anti-AQP4 antibody-negative patients than anti-AQP4 antibody-positive patients, approximately 30 to 50% of patients with anti-AQP4 antibody harbour brain MRI lesions indistinguishable from those present in typical MS patients, such as periventricular ovoid lesions, suggesting the existence of considerable overlap in brain MRI features between anti-AQP4 antibody-positive and -negative Asian patients. In the present study, NMO patients with brain lesions showed a significantly higher annualized relapse rate (P(corr)=0.017) and higher frequency of anti-AQP4 antibody (P(corr)<0.0001) than typical NMO patients without brain lesions, suggesting that development of brain lesions in NMO may reflect high disease activity and thus be a warning sign.

Genetic and Infectious Profiles of Japanese Multiple Sclerosis Patients

Background Nationwide surveys conducted in Japan over the past thirty years have revealed a four-fold increase in the estimated number of multiple sclerosis (MS) patients, a decrease in the age at onset, and successive increases in patients with conventional MS, which shows an involvement of multiple sites in the central nervous system, including the cerebrum and cerebellum. We aimed to clarify whether genetic and infectious backgrounds correlate to distinct disease phenotypes of MS in Japanese patients. Methodology/Principal Findings We analyzed HLA-DRB1 and -DPB1 alleles, and IgG antibodies specific for Helicobacter pylori, Chlamydia pneumoniae, varicella zoster virus, and Epstein-Barr virus nuclear antigen (EBNA) in 145 MS patients and 367 healthy controls (HCs). Frequencies of DRB1*0405 and DPB1*0301 were significantly higher, and DRB1*0901 and DPB1*0401 significantly lower, in MS patients as compared with HCs. MS patients with DRB1*0405 had a significantly earlier age of onset and lower Progression Index than patients without this allele. The proportion and absolute number of patients with DRB1*0405 successively increased with advancing year of birth. In MS patients without DRB1*0405, the frequency of the DRB1*1501 allele was significantly higher, while the DRB1*0901 allele was significantly lower, compared with HCs. Furthermore, DRB1*0405-negative MS patients were significantly more likely to be positive for EBNA antibodies compared with HCs. Conclusions Our study suggests that MS patients harboring DRB1*0405, a genetic risk factor for MS in the Japanese population, have a younger age at onset and a relatively benign disease course, while DRB1*0405-negative MS patients have features similar to Western-type MS in terms of association with Epstein-Barr virus infection and DRB1*1501. The recent increase of MS in young Japanese people may be caused, in part, by an increase in DRB1*0405-positive MS patients.

A nationwide survey of hypertrophic pachymeningitis in Japan

Objectives To clarify the prevalence, frequent causes and distinct features of hypertrophic pachymeningitis (HP) according to background conditions in a nationwide survey in Japan. Methods The study began with a preliminary survey to determine the approximate number of HP patients diagnosed from 1 January 2005 to 31 December 2009, and was followed by a questionnaire survey for clinical and laboratory findings. HP was defined as a condition with thickening of the cranial or spinal dura mater with inflammation, evidenced by MRI or histology. Results Crude HP prevalence was 0.949/100 000 population. The mean age at onset was 58.3±15.8 years. Among 159 cases for whom detailed data were collated, antineutrophil cytoplasmic antibody (ANCA)-related HP was found in 54 cases (34.0%) and IgG4/multifocal fibrosclerosis (MFS)-related HP in 14 cases (8.8%). Seventy cases (44.0%) were classified as ‘idiopathic’ and 21 (13.2%) as ‘others’. ANCA-related HP cases showed a female preponderance, a higher age of onset, and higher frequencies of otological symptoms and elevated systemic inflammatory biomarkers, but lower frequencies of diplopia compared with idiopathic HP. IgG4/MFS-related HP cases showed a marked male predominance; all had cranial HP while none had isolated spinal HP or decreased sensation. Conclusions HP is not extremely rare. ANCA-related HP is the most frequent form, followed by IgG4/MFS-related HP. Both forms have unique features, which may help to differentiate background causes.

Connexin 43 astrocytopathy linked to rapidly progressive multiple sclerosis and neuromyelitis optica.

Background Multiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO. Methods/Principal Findings Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, P=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, P=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients. Conclusions These findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.

Peripheral Blood T Cell Dynamics Predict Relapse in Multiple Sclerosis Patients on Fingolimod

Background Fingolimod efficiently reduces multiple sclerosis (MS) relapse by inhibiting lymphocyte egress from lymph nodes through down-modulation of sphingosine 1-phosphate (S1P) receptors. We aimed to clarify the alterations in peripheral blood T cell subsets associated with MS relapse on fingolimod. Methods/Principal Findings Blood samples successively collected from 23 relapsing-remitting MS patients before and during fingolimod therapy (0.5 mg/day) for 12 months and 18 healthy controls (HCs) were analysed for T cell subsets by flow cytometry. In MS patients, the percentages of central memory T (CCR7+CD45RO+) cells (TCM) and naïve T (CCR7+CD45RO-) cells decreased significantly, while those of effector memory T (CCR7-CD45RA-) and suppressor precursor T (CD28-) cells increased in both CD4+T and CD8+T cells from 2 weeks to 12 months during fingolimod therapy. The percentages of regulatory T (CD4+CD25highCD127low) cells in CD4+T cells and CCR7-CD45RA+T cells in CD8+T cells also increased significantly. Eight relapsed patients demonstrated greater percentages of CD4+TCM than 15 non-relapsed patients at 3 and 6 months (p=0.0051 and p=0.0088, respectively). The IL17-, IL9-, and IL4-producing CD4+T cell percentages were significantly higher at pre-treatment in MS patients compared with HCs (p<0.01 for all), while the IL17-producing CD4+T cell percentages tended to show a transient increase at 2 weeks of fingolimod therapy (pcorr=0.0834). Conclusions The CD4+TCM percentages at 2 weeks to 12 months during fingolimod therapy are related to relapse.

Extensive loss of connexins in Baló's disease: Evidence for an auto-antibody-independent astrocytopathy via impaired astrocyte-oligodendrocyte/ myelin interaction

Extensive aquaporin-4 (AQP4) loss without perivascular deposition of either activated complement or immunoglobulins is a characteristic of Baló’s disease. Our aim in this study was to investigate the relationship between astrocytopathy and demyelination in Baló’s disease, focusing on connexins (Cx), which form gap junctions among glial cells and myelin. Autopsied specimens from four cases that provided seven actively demyelinating concentric lesions infiltrated with numerous CD68+ macrophages were immunohistochemically examined for the astrocyte markers glial fibrillary acidic protein (GFAP), AQP4, Cx43, Cx30 and megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1). Specimens were also stained for oligodendrocyte/myelin markers, namely Cx32, Cx47, myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-specific protein (OSP) and Nogo-A. Serum samples from six patients that had undergone magnetic resonance imaging, confirming a diagnosis of Baló’s disease, were assayed for the presence of anti-Cx43, -Cx32 and -AQP4 antibodies. Despite the presence of numerous GFAP- and MLC1-positive astrocytes, there was a marked decrease in the levels of Cx43, Cx32 and Cx47. At the leading edges, Cx43 and AQP4 were mostly absent despite positive GFAP, MLC1, Cx32, Cx47, MOG, MAG, and OSP immunoreactivity. Of the six Baló’s disease patients, none were positive for anti-Cxs or -AQP4 antibodies. Baló’s disease is characterized by extensive loss of Cxs and AQP4, and a lack of auto-antibodies to Cxs and AQP4. Loss of Cx43 and AQP4 in the presence of other oligodendrocyte/myelin proteins at the leading edges suggests the possibility that auto-antibody-independent astrocytopathy may contribute to disease pathology via the disruption of astrocyte–oligodendrocyte/myelin interactions.

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.

Genetic and infectious profiles influence cerebrospinal fluid IgG abnormality in Japanese multiple sclerosis patients.

Background Abnormal intrathecal synthesis of IgG, reflected by cerebrospinal fluid (CSF) oligoclonal IgG bands (OBs) and increased IgG index, is much less frequently observed in Japanese multiple sclerosis (MS) cohorts compared with Western cohorts. We aimed to clarify whether genetic and common infectious backgrounds influence CSF IgG abnormality in Japanese MS patients. Methodology We analyzed HLA-DRB1 alleles, and IgG antibodies against Chlamydia pneumoniae, Helicobacter pylori, Epstein-Barr virus nuclear antigen (EBNA), and varicella zoster virus (VZV) in 94 patients with MS and 367 unrelated healthy controls (HCs). We defined CSF IgG abnormality as the presence of CSF OBs and/or increased IgG index (>0.658). Principal Findings CSF IgG abnormality was found in 59 of 94 (62.8%) MS patients. CSF IgG abnormality-positive patients had a significantly higher frequency of brain MRI lesions meeting the Barkhof criteria compared with abnormality-negative patients. Compared with HCs, CSF IgG abnormality-positive MS patients showed a significantly higher frequency of DRB1*1501, whereas CSF IgG abnormality-negative patients had a significantly higher frequency of DRB1*0405. CSF IgG abnormality-positive MS patients had a significantly higher frequency of anti-C. pneumoniae IgG antibodies compared with CSF IgG abnormality-negative MS patients, although there was no difference in the frequency of anti-C. pneumoniae IgG antibodies between HCs and total MS patients. Compared with HCs, anti-H. pylori IgG antibodies were detected significantly less frequently in the total MS patients, especially in CSF IgG abnormality-negative MS patients. The frequencies of antibodies against EBNA and VZV did not differ significantly among the groups. Conclusions CSF IgG abnormality is associated with Western MS-like brain MRI features. DRB1*1501 and C. pneumoniae infection confer CSF IgG abnormality, while DRB1*0405 and H. pylori infection are positively and negatively associated with CSF IgG abnormality-negative MS, respectively, suggesting that genetic and environmental factors differentially contribute to MS susceptibility according to the CSF IgG abnormality status.