Journal of Neuroimmunology 278 (2015) 26–29
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Quantitative analysis of aquaporin-4 antibody in longitudinally extensive transverse myelitis Jing Wang a, Xin Li a, Da-Qi Zhang a, Chun-Sheng Yang a, Yuan Qi a, Min-Shu Li a, Fu-Dong Shi a,b, Li Yang a,⁎ a b
Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Department of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ, USA
a r t i c l e
i n f o
Article history: Received 19 June 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online xxxx Keywords: Longitudinally extensive transverse myelitis Neuromyelitis optica spectrum disorders Aquaporin-4 antibody Fluorescence immunoprecipitation assay Clinical features
a b s t r a c t Aquaporin-4 (AQP-4) antibody-positive longitudinally extensive transverse myelitis (LETM) is referred to as a neuromyelitis optica (NMO) spectrum disorder. We conducted an exploratory investigation of correlations between AQP-4 antibody serum levels, as determined by a fluorescent immunoprecipitation assay, and clinical characteristics in LETM. Expanded Disability Status Scores (EDSS) scores and number of segments of spinal cord involved were positively correlated to AQP-4 antibody levels. However, serum AQP-4 antibody levels were not correlated with the time to next attack or the conversion time of LETM to NMO, although seropositive LETM patients demonstrated a high conversion rate to NMO (78.1%). © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Neuromyelitis optica (NMO) is an acute or subacute demyelinating illness, which predominantly affects the optic nerves and spinal cord. Longitudinally extensive transverse myelitis (LETM; i.e. myelitis, which extends over a continuous lesion at least 3 vertebral segments in length) is the hallmark of NMO (Tobin et al., 2014). Aquaporin-4 (AQP-4) antibody, or NMO-immunoglobulin G (IgG), is an important diagnostic biomarker of definite and limited forms of NMO, collectively referred to as NMO spectrum disorders (NMOSD; Lennon et al., 2004; Jarius and Wildemann, 2013). This classification now includes seropositive patients with attacks of only optic neuritis or LETM (Wingerchuk et al., 2007). AQP-4 IgG detection has become an essential tool in the diagnosis of patients at high risk for NMOSD (Waters et al., 2012). However, qualitative detection rather than quantitative determination of AQP-4 antibody has been most widely used in clinical research on NMOSD. The aim of the present study was to investigate clinical features associated with a range of different AQP-4 antibody levels.
2.1. Subjects
⁎ Corresponding author. E-mail addresses: [email protected] (J. Wang), [email protected] (X. Li), [email protected] (D.-Q. Zhang), [email protected] (C.-S. Yang), [email protected] (M.-S. Li), [email protected] (F.-D. Shi), [email protected] (L. Yang).
http://dx.doi.org/10.1016/j.jneuroim.2014.12.006 0165-5728/© 2014 Elsevier B.V. All rights reserved.
We tested 72 patients with first-ever LETM who were admitted to the Neuroimmunology Center of Tianjin Medical University General Hospital between January, 2008 and March, 2013. The LETM group was characterized by myelitis affecting ≥3 vertebral segments, termed longitudinally extensive spinal cord lesions (LESCL), with or without the brain lesions of NMO, based on the report of Pittock et al. (2006). At the last follow-up, 30 patients fulfilled the Wingerchuk diagnostic criteria for NMO (Wingerchuk et al., 2006); 28 patients were diagnosed as recurrent LETM, and the remaining 14 patients were still diagnosed as monophasic LETM. This study was approved by the Ethics Committee of our hospital and all patients gave their consent to participate in the study. Detailed clinical information and first episode blood samples were available from our database and the sample bank of Central Nervous System Demyelinating Disease, respectively. For detection of the AQP4 antibody, the patients' blood samples were collected during the acute phase before introducing immunosuppressive medicine (intravenous corticosteroids). The median latency between blood sampling and the onset of LETM was 4 days (range 1–7). Ten serum samples from healthy donors were obtained to comprise the healthy control group (HCs). Twenty serum samples from patients with neurological diseases other than NMOSD (Guillain–Barré syndrome, 6; cerebral vascular infarction, 7; and encephalitis, 7) were evaluated as the disease control
J. Wang et al. / Journal of Neuroimmunology 278 (2015) 26–29
group. EDSS scores were calculated twice — when the blood samples were collected and at the last follow-up. Spinal MRI scans were performed in the acute phase (1.5- or 3.0-Tesla) of the first episode. Spinal MRI abnormalities were defined as positive if they showed typical demyelinating lesions. The lengths of spinal cord lesions, the time to next attack and the conversion time of LETM to NMO were recorded. 2.2. Fluorescent immunoprecipitation assay (FIPA) for AQP-4 antibody testing Human embryonic kidney (HEK293) cells were planted in 175 cm2 flasks. When the adherent cells' confluence reached 50%, the cultured HEK293 cells were transfected with EGFP-tagged AQP4 M23 plasmid (donated by the Department of Clinical Neurology of Oxford University) using polyethyleneimine (PEI). After 48 h of incubation, the transfected cells were lysed with extraction buffer (10 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, with a protease inhibitor cocktail added immediately before use). The extracted EGFP-AQP-4 protein was acquired after centrifugation. The fluorescent units (FUs) were assayed and adjusted to 500–600 FU/200 μL extract. Twenty microliter serum was incubated with 200 μL extract at 4 °C overnight, after which the antibody IgG was precipitated by the addition of 75 μL Protein A Sepharose (previously blocked with ultra-low fetal calf serum). After thorough washing, the beads with 150 μL extraction buffer were removed to 96-well black microtiter plates and the FU values (Excitation 488 nm, Emission 507 nm) were obtained on a Microplate reader (SYNERGY 2, BioTek, U.S.A.; Waters and Vincent, 2008; Yang et al., 2011). Assays on positive samples above 400 FU were repeated by titrating the sera. In the FIPA, we established a cut-off value corresponding to the mean FU of the healthy control sera plus three standard deviations, such that the cut-off value for our AQP4-Ab detection was 7.8 FU. All samples were tested in the same run and duplicate samples were redetected by the same operator for evaluating intra-assay variation. Data analysis was performed on the average of the two results. Three samples (a negative patient sample, a low positive patient sample, and a high positive patient sample in a previous cell-based assay) were tested as above in 2 independent runs. The intrarun coefficients of variability (%CV) were 2.23 and 2.31. 2.3. Statistical analysis We used Spearman's rank correlation to explore potential correlations between AQP-4 antibody serum levels and clinical parameters such as EDSS scores, number of segments of spinal cord involved, time to next relapse, and conversion time of LETM to NMO. Correction for multiple testing was not performed due to the exploratory nature of this study. Student's t-test was used for between-group comparisons of AQP-4 antibody serum levels (with 10 at the bottom of the logarithmic transformation). Categorical variables were compared using the chi-squared test. The threshold for statistical significance was set at p b 0.05, α = 0.05. All statistical analyses were performed using GraphPad Prism Version 5.
27
Table 1 Demographic and clinical characteristics of 49 seropositive LETM patients. AQP-4 antibodypositive LETMn = 49 Sex (male/female) Age at onset (years), mean ± SD Disease duration (months), mean ± SD EDSS at sample collection time, mean ± SD EDSS at the last follow-up, mean ± SD Number of segments of spinal cord involved at the first episode, median (range) Number of brain lesions involved at the first episode, median (range) AQP-4 antibody serum levels at first LETM (FU), median (range) Time to next attack (irrespective of optic nerve, brain or spinal cord) (months), median (range) Conversion period of LETM to NMO (months), median (range)
7/42 47.10 ± 15.78 52.08 ± 22.67 6.03 ± 2.12 3.16 ± 1.44 6 (3,18) 1 (0,10) 56 (8,940) 12 (1,84) 21.5 (3,74)
AQP-4 — aquaporin-4; EDSS — expanded disability status scale; FU — fluorescent unit; LETM — longitudinally extensive transverse myelitis; NMO — neuromyelitis optica.
3.2. AQP-4 antibody levels and clinical characteristics in seropositive LETM patients The demographic and clinical features of the 49 AQP-4 antibodypositive patients can be summarized as follows. EDSS scores at the time of sample collection and at the last follow-up were both positively correlated with AQP-4 antibody levels (sample collection: r = 0.463, p = 0.001; last follow-up: r = 0.424, p = 0.003). The number of segments of spinal cord involved was weakly correlated with AQP-4 antibody levels (r = 0.142, p = 0.039). We made further comparisons of AQP-4 antibody levels according to the lesion length (10 segments) of spinal cord and found a significant difference (p b 0.05; Fig. 1). The time to next attack (irrespective of optic nerve, brain or spinal cord) was not correlated with AQP-4 antibody level (r = −0.29, p = 0.073; Fig. 2a, b, c, d). 3.3. Relationship of AQP-4 antibody status and conversion of LETM to NMO At the last follow-up visit, 30 patients of the total cohort (n = 72) had progressed to NMO (25/32 seropositive; 5/14 seronegative, p b 0.05; Fig. 3). The median time to NMO conversion was 21.5 months (range 3–74). Nevertheless, the conversion time of LETM to NMO was not correlated with AQP-4 antibody level at the first episode (r = −0.141, p = 0.458). (Fig. 2e). 4. Discussion In vitro studies have shown that NMO-IgG-mediated antibodydependent cellular cytotoxicity (ADCC) and complement-dependent
3. Results 3.1. Demographic, clinical, radiological and laboratory findings in patients with LETM Of the 72 patients who presented with idiopathic LETM, AQP-4 antibodies were detected in 49 patients such that the positivity for AQP-4 antibody was 68%. Twenty disease control patients were negative in the FIPA. Sensitivity and specificity were 68% (95% CI 62.5%–73.5%) and 100% (95% CI 83%–100%), respectively. Detailed characteristics of the 49 seropositive LETM patients are listed in Table 1. All 72 cases were followed up over a one-year period.
Fig. 1. Comparisons of AQP-4 antibody levels according to the lesion length (10 segments) of spinal cord. FU — fluorescent unit, LETM — longitudinally extensive transverse myelitis.
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J. Wang et al. / Journal of Neuroimmunology 278 (2015) 26–29
Fig. 2. Correlations between the AQP-4 antibody levels (FU) at first LETM and clinical parameters. a. EDSS scores at time of sample collection; b. EDSS scores at the last follow-up; c. Number of segments of spinal cord involved on MRI; d. The time to next attack (irrespective of optic nerve, brain or spinal cord); e. Conversion time of LETM to NMO; EDSS — expanded disability status scale; FU — fluorescent unit; LETM — longitudinally extensive transverse myelitis; NMO — neuromyelitis optica; M — month.
cytotoxicity (CDC) are important mechanisms mediating the death of astrocytes, such that NMO-IgG is thought to be involved in NMO pathogenesis (Saikali et al., 2009; Papadopoulos and Verkman, 2012; Jarius et al., 2014; Levy et al., 2014). Moreover, Takahashi reported that higher AQP-4 antibody titers were associated with complete blindness and more extensive spinal cord lesions on MRI (Takahashi et al., 2007). In contrast, others have shown that serostatus did not affect either attack
severity or disability outcome (Jiao et al., 2013). In the present study, we used FIPA to quantitatively detect NMO-IgG levels. The time for blood collection was matched with MRI examination and the patients, experiencing their first-ever LETM episode, did not receive immunosuppressive therapy before blood collection. The EDSS scores at the last visit, as an indicator of clinical outcome, were associated with the AQP-4 antibody levels at the first episode. This positive correlation
J. Wang et al. / Journal of Neuroimmunology 278 (2015) 26–29
29
Funding This work was supported by grants from the National Natural Science Foundation of China (81171183, 81471221 to L.Y.), the National Basic Research Program of China (2013CB966900 to F.D.S.) and the National Key Clinical Specialty Construction Program of China. Conflict of interest statement None of the authors on this study report conflicts of interest. Acknowledgments We thank Drs. A. Vincent and D. Beeson for providing plasmids for AQP4, and our patients for participating in this study. References
Fig. 3. Conversion time to NMO of seropositive (n = 25) and seronegative (n = 5) LETM patients. LETM — longitudinally extensive transverse myelitis; NMO — neuromyelitis optica; M — month.
indicates that NMO-IgG antibodies may be useful in predicting patients' final degree of disability. Studies on the conversion of LETM to NMO are relatively rare. Previous studies have shown that acute attacks are preceded by an elevation in AQP-4 antibody levels (Jarius et al., 2008). According to our findings, seropositive LETM patients displayed a high conversion rate to NMO (78.1%). These results indicate that AQP-4 antibody status may be of some prognostic relevance in NMOSD. However, there was no association between the time to next attack and AQP4-IgG levels. Whether AQP-4 antibody levels can likewise provide predictive support on the likelihood of relapse remains in question. It may be assumed that other inflammatory reactions, such as those mediated by T cells, complements, cytokines, or other autoantibodies, also participate in the conversion to NMO and may trigger the pathogenesis of the disease. We should monitor AQP-4 antibody concentrations and cellular immunity at follow-up periods to achieve improved possibilities for early prediction and prevention of more advanced disease states. There are some limitations to our study. First, due to the exploratory nature of this study, p values were not corrected for multiple comparisons. Second, the exploratory correlational analysis used in this study can only identify associations between variables, not the nature of cause and effect relationships. Together with our small study population and low number of controls, these conditions limited the power of our statistical analyses. Nevertheless, we believe that our results provide important evidence concerning the role of AQP-4 antibody in LETM and its potential progression to NMO.
Jarius, S., Wildemann, B., 2013. Aquaporin-4 antibodies (NMO-IgG) as a serological marker of neuromyelitis optica: a critical review of the literature. Brain Pathol. 23, 661–683. Jarius, S., Aboul-Enein, F., Waters, P., Kuenz, B., Hauser, A., Berger, T., Lang, W., Reindl, M., Vincent, A., Kristoferitsch, W., 2008. Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain 131, 3072–3080. Jarius, S., Wildemann, B., Paul, F., 2014. Neuromyelitis optica: clinical features, immunopathogenesis and treatment. Clin. Exp. Immunol. 176, 149–164. Jiao, Y., Fryer, J.P., Lennon, V.A., Jenkins, S.M., Quek, A.M., Smith, C.Y., McKeon, A., Costanzi, C., Iorio, R., Weinshenker, B.G., Wingerchuk, D.M., Shuster, E.A., Lucchinetti, C.F., Pittock, S.J., 2013. Updated estimate of AQP4-IgG serostatus and disability outcome in neuromyelitis optica. Neurology 81, 1197–1204. Lennon, V.A., Wingerchuk, D.M., Kryzer, T.J., Pittock, S.J., Lucchinetti, C.F., Fujihara, K., Nakashima, I., Weinshenker, B.G., 2004. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 364, 2106–2112. Levy, M., Wildemann, B., Jarius, S., Orellano, B., Sasidharan, S., Weber, M.S., Stuve, O., 2014. Immunopathogenesis of neuromyelitis optica. Adv. Immunol. 121, 213–242. Papadopoulos, M.C., Verkman, A.S., 2012. Aquaporin 4 and neuromyelitis optica. Lancet Neurol. 11, 535–544. Pittock, S.J., Lennon, V.A., Krecke, K., Wingerchuk, D.M., Lucchinetti, C.F., Weinshenker, B.G., 2006. Brain abnormalities in neuromyelitis optica. Arch. Neurol. 63, 390–396. Saikali, P., Cayrol, R., Vincent, T., 2009. Anti-aquaporin-4 auto-antibodies orchestrate the pathogenesis in neuromyelitis optica. Autoimmun. Rev. 9, 132–135. Takahashi, T., Fujihara, K., Nakashima, I., Misu, T., Miyazawa, I., Nakamura, M., Watanabe, S., Shiga, Y., Kanaoka, C., Fujimori, J., Sato, S., Itoyama, Y., 2007. Anti-aquaporin-4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain 130, 1235–1243. Tobin, W.O., Weinshenker, B.G., Lucchinetti, C.F., 2014. Longitudinally extensive transverse myelitis. Curr. Opin. Neurol. 27, 279–289. Waters, P., Vincent, A., 2008. Detection of anti-aquaporin-4 antibodies in neuromyelitis optica: current status of the assays. Int. M.S. J. 15, 99–105. Waters, P.J., McKeon, A., Leite, M.I., Rajasekharan, S., Lennon, V.A., Villalobos, A., Palace, J., Mandrekar, J.N., Vincent, A., Bar-Or, A., Pittock, S.J., 2012. Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays. Neurology 78, 665–671. Wingerchuk, D.M., Lennon, V.A., Pittock, S.J., Lucchinetti, C.F., Weinshenker, B.G., 2006. Revised diagnostic criteria for neuromyelitis optica. Neurology 66, 1485–1489. Wingerchuk, D.M., Lennon, V.A., Lucchinetti, C.F., Pittock, S.J., Weinshenker, B.G., 2007. The spectrum of neuromyelitis optica. Lancet Neurol. 6, 805–815. Yang, L., Maxwell, S., Leite, M.I., Waters, P., Clover, L., Fan, X., Zhang, D., Yang, C., Beeson, D., Vincent, A., 2011. Non-radioactive serological diagnosis of myasthenia gravis and clinical features of patients from Tianjin. Chin. J. Neurol. Sci. 301, 71–76.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Quantitative analysis of aquaporin-4 antibody in longitudinally extensive transverse myelitis Jing Wang a, Xin Li a, Da-Qi Zhang a, Chun-Sheng Yang a, Yuan Qi a, Min-Shu Li a, Fu-Dong Shi a,b, Li Yang a,⁎ a b
Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Department of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ, USA
a r t i c l e
i n f o
Article history: Received 19 June 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online xxxx Keywords: Longitudinally extensive transverse myelitis Neuromyelitis optica spectrum disorders Aquaporin-4 antibody Fluorescence immunoprecipitation assay Clinical features
a b s t r a c t Aquaporin-4 (AQP-4) antibody-positive longitudinally extensive transverse myelitis (LETM) is referred to as a neuromyelitis optica (NMO) spectrum disorder. We conducted an exploratory investigation of correlations between AQP-4 antibody serum levels, as determined by a fluorescent immunoprecipitation assay, and clinical characteristics in LETM. Expanded Disability Status Scores (EDSS) scores and number of segments of spinal cord involved were positively correlated to AQP-4 antibody levels. However, serum AQP-4 antibody levels were not correlated with the time to next attack or the conversion time of LETM to NMO, although seropositive LETM patients demonstrated a high conversion rate to NMO (78.1%). © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Neuromyelitis optica (NMO) is an acute or subacute demyelinating illness, which predominantly affects the optic nerves and spinal cord. Longitudinally extensive transverse myelitis (LETM; i.e. myelitis, which extends over a continuous lesion at least 3 vertebral segments in length) is the hallmark of NMO (Tobin et al., 2014). Aquaporin-4 (AQP-4) antibody, or NMO-immunoglobulin G (IgG), is an important diagnostic biomarker of definite and limited forms of NMO, collectively referred to as NMO spectrum disorders (NMOSD; Lennon et al., 2004; Jarius and Wildemann, 2013). This classification now includes seropositive patients with attacks of only optic neuritis or LETM (Wingerchuk et al., 2007). AQP-4 IgG detection has become an essential tool in the diagnosis of patients at high risk for NMOSD (Waters et al., 2012). However, qualitative detection rather than quantitative determination of AQP-4 antibody has been most widely used in clinical research on NMOSD. The aim of the present study was to investigate clinical features associated with a range of different AQP-4 antibody levels.
2.1. Subjects
⁎ Corresponding author. E-mail addresses: [email protected] (J. Wang), [email protected] (X. Li), [email protected] (D.-Q. Zhang), [email protected] (C.-S. Yang), [email protected] (M.-S. Li), [email protected] (F.-D. Shi), [email protected] (L. Yang).
http://dx.doi.org/10.1016/j.jneuroim.2014.12.006 0165-5728/© 2014 Elsevier B.V. All rights reserved.
We tested 72 patients with first-ever LETM who were admitted to the Neuroimmunology Center of Tianjin Medical University General Hospital between January, 2008 and March, 2013. The LETM group was characterized by myelitis affecting ≥3 vertebral segments, termed longitudinally extensive spinal cord lesions (LESCL), with or without the brain lesions of NMO, based on the report of Pittock et al. (2006). At the last follow-up, 30 patients fulfilled the Wingerchuk diagnostic criteria for NMO (Wingerchuk et al., 2006); 28 patients were diagnosed as recurrent LETM, and the remaining 14 patients were still diagnosed as monophasic LETM. This study was approved by the Ethics Committee of our hospital and all patients gave their consent to participate in the study. Detailed clinical information and first episode blood samples were available from our database and the sample bank of Central Nervous System Demyelinating Disease, respectively. For detection of the AQP4 antibody, the patients' blood samples were collected during the acute phase before introducing immunosuppressive medicine (intravenous corticosteroids). The median latency between blood sampling and the onset of LETM was 4 days (range 1–7). Ten serum samples from healthy donors were obtained to comprise the healthy control group (HCs). Twenty serum samples from patients with neurological diseases other than NMOSD (Guillain–Barré syndrome, 6; cerebral vascular infarction, 7; and encephalitis, 7) were evaluated as the disease control
J. Wang et al. / Journal of Neuroimmunology 278 (2015) 26–29
group. EDSS scores were calculated twice — when the blood samples were collected and at the last follow-up. Spinal MRI scans were performed in the acute phase (1.5- or 3.0-Tesla) of the first episode. Spinal MRI abnormalities were defined as positive if they showed typical demyelinating lesions. The lengths of spinal cord lesions, the time to next attack and the conversion time of LETM to NMO were recorded. 2.2. Fluorescent immunoprecipitation assay (FIPA) for AQP-4 antibody testing Human embryonic kidney (HEK293) cells were planted in 175 cm2 flasks. When the adherent cells' confluence reached 50%, the cultured HEK293 cells were transfected with EGFP-tagged AQP4 M23 plasmid (donated by the Department of Clinical Neurology of Oxford University) using polyethyleneimine (PEI). After 48 h of incubation, the transfected cells were lysed with extraction buffer (10 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, with a protease inhibitor cocktail added immediately before use). The extracted EGFP-AQP-4 protein was acquired after centrifugation. The fluorescent units (FUs) were assayed and adjusted to 500–600 FU/200 μL extract. Twenty microliter serum was incubated with 200 μL extract at 4 °C overnight, after which the antibody IgG was precipitated by the addition of 75 μL Protein A Sepharose (previously blocked with ultra-low fetal calf serum). After thorough washing, the beads with 150 μL extraction buffer were removed to 96-well black microtiter plates and the FU values (Excitation 488 nm, Emission 507 nm) were obtained on a Microplate reader (SYNERGY 2, BioTek, U.S.A.; Waters and Vincent, 2008; Yang et al., 2011). Assays on positive samples above 400 FU were repeated by titrating the sera. In the FIPA, we established a cut-off value corresponding to the mean FU of the healthy control sera plus three standard deviations, such that the cut-off value for our AQP4-Ab detection was 7.8 FU. All samples were tested in the same run and duplicate samples were redetected by the same operator for evaluating intra-assay variation. Data analysis was performed on the average of the two results. Three samples (a negative patient sample, a low positive patient sample, and a high positive patient sample in a previous cell-based assay) were tested as above in 2 independent runs. The intrarun coefficients of variability (%CV) were 2.23 and 2.31. 2.3. Statistical analysis We used Spearman's rank correlation to explore potential correlations between AQP-4 antibody serum levels and clinical parameters such as EDSS scores, number of segments of spinal cord involved, time to next relapse, and conversion time of LETM to NMO. Correction for multiple testing was not performed due to the exploratory nature of this study. Student's t-test was used for between-group comparisons of AQP-4 antibody serum levels (with 10 at the bottom of the logarithmic transformation). Categorical variables were compared using the chi-squared test. The threshold for statistical significance was set at p b 0.05, α = 0.05. All statistical analyses were performed using GraphPad Prism Version 5.
27
Table 1 Demographic and clinical characteristics of 49 seropositive LETM patients. AQP-4 antibodypositive LETMn = 49 Sex (male/female) Age at onset (years), mean ± SD Disease duration (months), mean ± SD EDSS at sample collection time, mean ± SD EDSS at the last follow-up, mean ± SD Number of segments of spinal cord involved at the first episode, median (range) Number of brain lesions involved at the first episode, median (range) AQP-4 antibody serum levels at first LETM (FU), median (range) Time to next attack (irrespective of optic nerve, brain or spinal cord) (months), median (range) Conversion period of LETM to NMO (months), median (range)
7/42 47.10 ± 15.78 52.08 ± 22.67 6.03 ± 2.12 3.16 ± 1.44 6 (3,18) 1 (0,10) 56 (8,940) 12 (1,84) 21.5 (3,74)
AQP-4 — aquaporin-4; EDSS — expanded disability status scale; FU — fluorescent unit; LETM — longitudinally extensive transverse myelitis; NMO — neuromyelitis optica.
3.2. AQP-4 antibody levels and clinical characteristics in seropositive LETM patients The demographic and clinical features of the 49 AQP-4 antibodypositive patients can be summarized as follows. EDSS scores at the time of sample collection and at the last follow-up were both positively correlated with AQP-4 antibody levels (sample collection: r = 0.463, p = 0.001; last follow-up: r = 0.424, p = 0.003). The number of segments of spinal cord involved was weakly correlated with AQP-4 antibody levels (r = 0.142, p = 0.039). We made further comparisons of AQP-4 antibody levels according to the lesion length (10 segments) of spinal cord and found a significant difference (p b 0.05; Fig. 1). The time to next attack (irrespective of optic nerve, brain or spinal cord) was not correlated with AQP-4 antibody level (r = −0.29, p = 0.073; Fig. 2a, b, c, d). 3.3. Relationship of AQP-4 antibody status and conversion of LETM to NMO At the last follow-up visit, 30 patients of the total cohort (n = 72) had progressed to NMO (25/32 seropositive; 5/14 seronegative, p b 0.05; Fig. 3). The median time to NMO conversion was 21.5 months (range 3–74). Nevertheless, the conversion time of LETM to NMO was not correlated with AQP-4 antibody level at the first episode (r = −0.141, p = 0.458). (Fig. 2e). 4. Discussion In vitro studies have shown that NMO-IgG-mediated antibodydependent cellular cytotoxicity (ADCC) and complement-dependent
3. Results 3.1. Demographic, clinical, radiological and laboratory findings in patients with LETM Of the 72 patients who presented with idiopathic LETM, AQP-4 antibodies were detected in 49 patients such that the positivity for AQP-4 antibody was 68%. Twenty disease control patients were negative in the FIPA. Sensitivity and specificity were 68% (95% CI 62.5%–73.5%) and 100% (95% CI 83%–100%), respectively. Detailed characteristics of the 49 seropositive LETM patients are listed in Table 1. All 72 cases were followed up over a one-year period.
Fig. 1. Comparisons of AQP-4 antibody levels according to the lesion length (10 segments) of spinal cord. FU — fluorescent unit, LETM — longitudinally extensive transverse myelitis.
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J. Wang et al. / Journal of Neuroimmunology 278 (2015) 26–29
Fig. 2. Correlations between the AQP-4 antibody levels (FU) at first LETM and clinical parameters. a. EDSS scores at time of sample collection; b. EDSS scores at the last follow-up; c. Number of segments of spinal cord involved on MRI; d. The time to next attack (irrespective of optic nerve, brain or spinal cord); e. Conversion time of LETM to NMO; EDSS — expanded disability status scale; FU — fluorescent unit; LETM — longitudinally extensive transverse myelitis; NMO — neuromyelitis optica; M — month.
cytotoxicity (CDC) are important mechanisms mediating the death of astrocytes, such that NMO-IgG is thought to be involved in NMO pathogenesis (Saikali et al., 2009; Papadopoulos and Verkman, 2012; Jarius et al., 2014; Levy et al., 2014). Moreover, Takahashi reported that higher AQP-4 antibody titers were associated with complete blindness and more extensive spinal cord lesions on MRI (Takahashi et al., 2007). In contrast, others have shown that serostatus did not affect either attack
severity or disability outcome (Jiao et al., 2013). In the present study, we used FIPA to quantitatively detect NMO-IgG levels. The time for blood collection was matched with MRI examination and the patients, experiencing their first-ever LETM episode, did not receive immunosuppressive therapy before blood collection. The EDSS scores at the last visit, as an indicator of clinical outcome, were associated with the AQP-4 antibody levels at the first episode. This positive correlation
J. Wang et al. / Journal of Neuroimmunology 278 (2015) 26–29
29
Funding This work was supported by grants from the National Natural Science Foundation of China (81171183, 81471221 to L.Y.), the National Basic Research Program of China (2013CB966900 to F.D.S.) and the National Key Clinical Specialty Construction Program of China. Conflict of interest statement None of the authors on this study report conflicts of interest. Acknowledgments We thank Drs. A. Vincent and D. Beeson for providing plasmids for AQP4, and our patients for participating in this study. References
Fig. 3. Conversion time to NMO of seropositive (n = 25) and seronegative (n = 5) LETM patients. LETM — longitudinally extensive transverse myelitis; NMO — neuromyelitis optica; M — month.
indicates that NMO-IgG antibodies may be useful in predicting patients' final degree of disability. Studies on the conversion of LETM to NMO are relatively rare. Previous studies have shown that acute attacks are preceded by an elevation in AQP-4 antibody levels (Jarius et al., 2008). According to our findings, seropositive LETM patients displayed a high conversion rate to NMO (78.1%). These results indicate that AQP-4 antibody status may be of some prognostic relevance in NMOSD. However, there was no association between the time to next attack and AQP4-IgG levels. Whether AQP-4 antibody levels can likewise provide predictive support on the likelihood of relapse remains in question. It may be assumed that other inflammatory reactions, such as those mediated by T cells, complements, cytokines, or other autoantibodies, also participate in the conversion to NMO and may trigger the pathogenesis of the disease. We should monitor AQP-4 antibody concentrations and cellular immunity at follow-up periods to achieve improved possibilities for early prediction and prevention of more advanced disease states. There are some limitations to our study. First, due to the exploratory nature of this study, p values were not corrected for multiple comparisons. Second, the exploratory correlational analysis used in this study can only identify associations between variables, not the nature of cause and effect relationships. Together with our small study population and low number of controls, these conditions limited the power of our statistical analyses. Nevertheless, we believe that our results provide important evidence concerning the role of AQP-4 antibody in LETM and its potential progression to NMO.
Jarius, S., Wildemann, B., 2013. Aquaporin-4 antibodies (NMO-IgG) as a serological marker of neuromyelitis optica: a critical review of the literature. Brain Pathol. 23, 661–683. Jarius, S., Aboul-Enein, F., Waters, P., Kuenz, B., Hauser, A., Berger, T., Lang, W., Reindl, M., Vincent, A., Kristoferitsch, W., 2008. Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain 131, 3072–3080. Jarius, S., Wildemann, B., Paul, F., 2014. Neuromyelitis optica: clinical features, immunopathogenesis and treatment. Clin. Exp. Immunol. 176, 149–164. Jiao, Y., Fryer, J.P., Lennon, V.A., Jenkins, S.M., Quek, A.M., Smith, C.Y., McKeon, A., Costanzi, C., Iorio, R., Weinshenker, B.G., Wingerchuk, D.M., Shuster, E.A., Lucchinetti, C.F., Pittock, S.J., 2013. Updated estimate of AQP4-IgG serostatus and disability outcome in neuromyelitis optica. Neurology 81, 1197–1204. Lennon, V.A., Wingerchuk, D.M., Kryzer, T.J., Pittock, S.J., Lucchinetti, C.F., Fujihara, K., Nakashima, I., Weinshenker, B.G., 2004. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 364, 2106–2112. Levy, M., Wildemann, B., Jarius, S., Orellano, B., Sasidharan, S., Weber, M.S., Stuve, O., 2014. Immunopathogenesis of neuromyelitis optica. Adv. Immunol. 121, 213–242. Papadopoulos, M.C., Verkman, A.S., 2012. Aquaporin 4 and neuromyelitis optica. Lancet Neurol. 11, 535–544. Pittock, S.J., Lennon, V.A., Krecke, K., Wingerchuk, D.M., Lucchinetti, C.F., Weinshenker, B.G., 2006. Brain abnormalities in neuromyelitis optica. Arch. Neurol. 63, 390–396. Saikali, P., Cayrol, R., Vincent, T., 2009. Anti-aquaporin-4 auto-antibodies orchestrate the pathogenesis in neuromyelitis optica. Autoimmun. Rev. 9, 132–135. Takahashi, T., Fujihara, K., Nakashima, I., Misu, T., Miyazawa, I., Nakamura, M., Watanabe, S., Shiga, Y., Kanaoka, C., Fujimori, J., Sato, S., Itoyama, Y., 2007. Anti-aquaporin-4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain 130, 1235–1243. Tobin, W.O., Weinshenker, B.G., Lucchinetti, C.F., 2014. Longitudinally extensive transverse myelitis. Curr. Opin. Neurol. 27, 279–289. Waters, P., Vincent, A., 2008. Detection of anti-aquaporin-4 antibodies in neuromyelitis optica: current status of the assays. Int. M.S. J. 15, 99–105. Waters, P.J., McKeon, A., Leite, M.I., Rajasekharan, S., Lennon, V.A., Villalobos, A., Palace, J., Mandrekar, J.N., Vincent, A., Bar-Or, A., Pittock, S.J., 2012. Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays. Neurology 78, 665–671. Wingerchuk, D.M., Lennon, V.A., Pittock, S.J., Lucchinetti, C.F., Weinshenker, B.G., 2006. Revised diagnostic criteria for neuromyelitis optica. Neurology 66, 1485–1489. Wingerchuk, D.M., Lennon, V.A., Lucchinetti, C.F., Pittock, S.J., Weinshenker, B.G., 2007. The spectrum of neuromyelitis optica. Lancet Neurol. 6, 805–815. Yang, L., Maxwell, S., Leite, M.I., Waters, P., Clover, L., Fan, X., Zhang, D., Yang, C., Beeson, D., Vincent, A., 2011. Non-radioactive serological diagnosis of myasthenia gravis and clinical features of patients from Tianjin. Chin. J. Neurol. Sci. 301, 71–76.
