2010 11 4 11 Chin J CliniciansElectronic EditionNovember 15 2010 Vol. 4 No. 11 2147 NMO-IgG /anti-aqp4 NMO-IgG /anti-aqp4 NMO CNS TBM NMO-IgG /anti-aqp4 NMO NMO-IgG IIF AQP4-Ab CBA IIF NMO-IgG NMO-IgG AQP4-Ab CBA AQP4-Ab 172 NMO 24 LETM22 MS30 TBM 46 30 20 NMO-IgG /anti-aqp4 NMO TBM NMO-IgG TBM 76. 1% 35 /46 NMO 62. 5% 15 /24 LETM 59. 1% 13 /22 TBM NMO LETM CBA TBM 91. 3% 42 /46 NMO 91. 7% 22 /24 LETM 86. 4% 19 /22 MS CNS NMO-IgG /anti-aqp4 NMO-IgG /AQP4-Ab CNS 4 Detection of NMO-IgG or anti-aqp4 antibodiesa specifity challenge SUN Qiao-songLIU Jun-xiu FENG Yan-qingFANG Yi-minGUO NingLAI RongCHEN XiHUANG Fan. Department of Neurology First Affiliated Hospital of SUN Yat-sen UniversityGuangzhou 510080China Corresponding authorfeng Yan-qingEmailfyqgz@ vip. sina. com Abstract Objective Antibody against aquaporin-4 AQP4is a validated specific biomarker for the neuromyelitis optica NMOspectrum 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 TBMand 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 assaybased on indirect immunofluorescent IIF reactivity with mouse brain cryosectionsand an AQP4-transfected cell-based assay CBA. IIF we characterized further by dual immunostainingand 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 LETM22 multiple sclerosis MS30 pulmonary tuberculosis PTB30 tuberculosis meningitis TBM46 and healthy controls HC20. 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 patientsand titers of DOI10. 3877 /cma. j. issn. 1674-0785. 2010. 11. 017 B2009056 2004B33801006 510080 Emailfyqgz@ vip. sina. com
2148 2010 11 4 11 Chin J CliniciansElectronic EditionNovember 15 2010 Vol. 4 No. 11 anti-aqp4 IgG in TBM patients were higher than that in those with confirmed NMO and LTEM. Using the IIF assayseropositivity rates for NMO-IgG were 76. 1% 35 /46for TBM patients62. 5% 15 /24for NMO patientsand 59. 1% 13 /22for LETM. Using cell-based assays we found antibodies to AQP4 in 91. 3% 42 /46of TBM patients91. 7% 22 /24for NMO patientsand 86. 4% 19 /22for LETM. PTBMSand 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 antibodyand suggests NMO-IgG / anti-aqp4 may be a marker of destructive demyelination. Key words Neuromyelitis optica Meningitis bacterial Aquaporin 4 Antibody specificity 2004 Lennon 1 IIF NMO IgG NMO-IgG 2005 Lennon 2 NMO-IgG AQP4NMO-IgG AQP4-Ab NMO-IgG NMO OSMS LETM NMO 3 AQP4-Ab Takahashi 4 AQP4 HEK-293 AQP4 CBA IIF NMO-IgG / AQP4-Ab NMO MS 2006 Wingerchuk 5 NMO NMO-IgG /anti-aqp4 6 NMO 7 NMO MTB 8-9 TBM NMO 1. 1 2009 2 2010 2 152 24 NMO 22 LETM 30 MS 46 TBM 30 20 NMO 2006 Wingerchuk 5 NMO-IgG LETM NMO 3 MS McDonald 10 5 19 6 TBM 1 2 50 ~ 500 3 4 MTB MTB X 2. IIF NMO-IgG Lennon 1 NMO-IgG C57 18 ~ 25 g OCT 10 μm 30 min 1 60 1 604 FITC IgG1 100 PBS AQP4-Ab AQP4 1 200 2 h FITC IgG 1 100CY3 IgG 1 100 1 h
2010 11 4 11 Chin J CliniciansElectronic EditionNovember 15 2010 Vol. 4 No. 11 2149 3. CBA AQP4 Matsuoka 11 AQP4 QIAGEN RNA RNA RT-PCR cdna AQP4 M1 M23 PCR M1 5'-CTAGCTAGCATGAGTGACAGACCCACAG-3' M23 5'-CTAGCTAGCATGGTGGCTTTCAAAGGGGTC-3' 5'-GAAGATCTTACTGAAGA- CAATACCTCT-3' M1 M23 pegfp-n3 pegfp-n3-aqp4 HEK293 G418 AQP4 EGFP /HEK293 AQP4 EGFP-AQP4 /HEK293 EGFP /HEK293 24 h 10% 5 min PBS 1 h 1 604 CY3 IgG1 100 1 h PBS 4. SPSS 13. 0 Fisher Mann-Whitney P < 0. 05 1. NMO-IgG 24 NMO 15 62. 5% 22 LETM 13 59. 1% 46 TBM 35 76. 1% 30 PTB2 6. 7% 30 MS 1 3. 3% 20 HC NMO-IgG1 NMO-IgG AQP4 2A NMO TBM LETM NMO-IgG 2B Mann-Whitney TBM NMOP = 0. 034 LETM P = 0. 002 P < 0. 05 NMO LETM P = 0. 269 TBM NMO-IgG NMO LETM NMO LETM 2. AQP4 EGFP-AQP4 /HEK293 AQP4-Ab
2150 2010 11 4 11 Chin J CliniciansElectronic EditionNovember 15 2010 Vol. 4 No. 11 420 ~ 490 nm GFP EGFP /HEK293 EGFP CY3 IgG 535 ~ 550 nm IIF AQP4 CY3 GFP AQP4 3 24 NMO 22 91. 7% 22 LETM 19 86. 4% 46 TBM 42 91. 3% 30 PTB1 3. 3% 30 1 3. 3% 20 AQP4 1 Fisher NMO TBM LETM Fisher NMO P = 0. 036 1 172 NMO-IgG /anti-aqp4 NMO-IgGIIF % AQP4-AbCBA % AQP4-Ab TBM 46 1 1. 2 32. 718 ~ 65 3576. 1 4291. 3 NMO 24 1 7 3614 ~ 50 1562. 5 2291. 7 LETM 22 1 2. 5 339 ~ 53 1359. 1 1986. 4 30 1 2. 8 4017 ~ 65 13. 3 13. 3 30 1 4 39. 120 ~ 70 26. 7 13. 3 20 1 1 26. 221 ~ 43 00 00 NMO-IgG /AQP4-Ab TBM NMO-IgG /AQP4-Ab NMO NMO MS NMO-IgG /AQP4-Ab 1 12 NMO MS TBM NMO-IgG /AQP4-Ab
2010 11 4 11 Chin J CliniciansElectronic EditionNovember 15 2010 Vol. 4 No. 11 2151 NMO-IgG /anti-aqp4 IIF AQP4 IIF CBA NMO-IgG NMO-IgG AQP4 AQP4 CBA AQP4-cDNA HEK-293 NMO-IgG CBA NMO AQP4 HEK293 13 1 NMO NMO-IgG AQP4-Ab FIPA ELSA RIPA 13 NMO-IgG /AQP4-Ab NMO MS NMO-IgG 1 AQP4 14 Wingerchuk 2006 NMO 60% NMO MRI 15 NMO NMO-IgG / AQP4-Ab NMO 16 TBM AQP4-Ab NMO TBM NMO-IgG /AQP4-Ab TBM MTB AQP4 Kinoshita 17 NMO NMO
2152 2010 11 4 11 Chin J CliniciansElectronic EditionNovember 15 2010 Vol. 4 No. 11 AQP4-Ab 400 μg H37Ra MTB MTB 18 MTB 30 NMO-IgG /AQP4 TBM TBM NMO-IgG /AQP4 NMO-IgG /AQP4 NMO NMO 1Lennon VAWingerchuk DMKryzer Lancet 2004 36494512106-2112. TJet al. A serum autoantibody marker of neuromyelitis opticadistinction from multiple sclerosis. 2Lennon VA Kryzer TJ Pittock SJ et al. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005 2024 473-477. 3Wingerchuk DM Lennon VA Lucchinetti CF et al. The spectrum of neuromyelitis optica. Lancet Neurol 2007 69 805-815. 4Takahashi T Fujihara K Nakashima I et al. Establishment of a new sensitive assay for anti-human aquaporin-4 antibody in neuromyelitis optica. Tohoku J Exp Med 2006 2104 307-313. 5Wingerchuk DM Lennon VA Pittock SJ et al. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006 66101485-1489. 6Weinshenker BG. Clinical overview of neuromyelitis optica. Rinsho Shinkeigaku 2009 4911894-895. 7Pittock SJLennon VA. Aquaporin-4 autoantibodies in a paraneoplastic context. Arch Neurol 2008 655 629-632. 8Feng YQ Guo N Huang F et al. Anti-tuberculosis treatment for Devic's neuromyelitis optica. J Clin Neurosci 2010 17111372-1377. 9. NMO 13. 2008 885 310-313. 10McDonald WI Compston A Edan G et al. Recommended diagnostic criteria for multiple sclerosisguidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001 501 121-127. 11Matsuoka TMatsushita TKawano Y et al. Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesionsin multiple sclerosis in Japanese. Brain 2007 130Pt 51206-1223. 12Waters PJarius Neurol 2008 657 913-919. SLittleton E et al. Aquaporin-4 antibodies in neuromyelitis optica and longitudinally extensive transverse myelitis. Arch 13Fazio RMalosio MLLampasona V et al. Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler 2009 15101153-1163. 14Saiki S Ueno YMoritani T et al. Extensive hemispheric lesions with radiological evidence of blood-brain barrier integrity in a patient with neuromyelitis optica. J Neurol Sci 2009 2841 /2217-219. 15Pittock SJ Lennon VA Krecke K et al. Brain abnormalities in neuromyelitis optica. Arch Neurol 2006 633 390-396. 16Nandhagopal R Al-Asmi A Gujjar AR. Neuromyelitis opticaan overview. Postgrad Med J 2010 861013153-159. 17Kinoshita MNakatsuji YKimura T et al. Neuromyelitis opticapassive transfer to rats by human immunoglobulin. Biochem Biophys Res Commun 2009 3864 623-627. 18Murase TZheng RBJoe M et al. Structural insights into antibody recognition of mycobacterial polysaccharides. J Mol Biol 2009 3922 381-392. 2010-05-05. NMO-IgG /anti-aqp4 J /CD. 2010 411 2147-2152.