0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Original Contribution |

Quantification and Functional Characterization of Antibodies to Native Aquaporin 4 in Neuromyelitis Optica FREE

Sudhakar Reddy Kalluri, MSc; Zsolt Illes, MD; Rajneesh Srivastava, MSc; Bruce Cree, MD; Til Menge, MD; Jeffrey L. Bennett, MD, PhD; Achim Berthele, MD; Bernhard Hemmer, MD
[+] Author Affiliations

Author Affiliations: Department of Neurology, Klinikum rechts der Isar, Technische Universität München, München, Germany (Messrs Kalluri and Srivastava and Drs Berthele and Hemmer); Department of Neurology, University of Pecs, Pecs, Hungary (Dr Illes); Department of Neurology, University of California, San Francisco (Dr Cree); Department of Neurology, University of Düsseldorf, Düsseldorf, Germany (Dr Menge); and Departments of Neurology and Ophthalmology, University of Colorado Denver, Aurora (Dr Bennett).


Arch Neurol. 2010;67(10):1201-1208. doi:10.1001/archneurol.2010.269.
Text Size: A A A
Published online

Background  Antibodies targeting membrane proteins play an important role in various autoimmune diseases of the nervous system. So far, assays allowing proper analysis of such autoantibodies are largely missing. A serum autoantibody to aquaporin 4 (AQP4) is associated with neuromyelitis optica (NMO). Although several assays are able to detect this autoantibody, they do not allow determination of the biological activity of anti-AQP4 antibodies.

Objective  To develop a bioassay for quantification and characterization of human anti-AQP4 antibodies.

Design, Setting, and Participants  We developed a novel bioassay for quantification and characterization of human anti-AQP4 antibodies based on high-level expression of native AQP4 (nAQP4) protein on the surface of human astroglioma cells. The test was validated in 2 independent cohorts of patients with NMO spectrum disease.

Results  We detected anti-nAQP4-IgG with a sensitivity of 57.9% and specificity of 100% in patients with NMO spectrum diseases, suggesting that our bioassay is at least as sensitive and specific as the gold-standard NMO-IgG assay. The anti-AQP4 antibodies belonged predominantly to the IgG1 isotype and bound with high affinity to the extracellular domain of nAQP4. Our data suggest that the autoantibody exerts pathological properties because nAQP4-IgG–positive sera induced cell death of nAQP4-expressing cells by antibody-dependent cellular natural killer cell cytotoxic effect and complement activation. Furthermore, nAQP4-IgG titers strongly correlated with in vitro cytotoxic effect.

Conclusions  In NMO, this assay may help to unravel the biological function of anti-nAQP4-IgG. Our findings demonstrate the potential of bioassays to characterize biologically relevant antibodies in human autoimmune diseases.

Figures in this Article

Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system (CNS) that predominantly affects the optic nerves and spinal cord.1,2 The spectrum of NMO-related diseases also includes relapsing inflammatory optic neuritis (RION) and longitudinally extensive transverse myelitis (LETM). In most patients a specific serum autoantibody is found (NMO-IgG), which targets aquaporin 4 (AQP4), a water channel predominantly expressed on astrocytes in the spinal cord, optic nerve, and periventricular hypothalamic area.3,4 Several clinicopathological correlations have implicated NMO-IgG in NMO pathogenesis. First, a marked loss of AQP4 is observed in CNS lesions of NMO-IgG–positive patients.5 Second, the anti-AQP4 antibody titers correlate with the extent of spinal lesions at the nadir of NMO exacerbations.6 Third, serum anti-AQP4 antibody titers correlate with clinical disease activity.7 Recently, anti-AQP4 antibodies were shown to activate complement,8,9 induce degranulation of natural killer (NK) cells,10 disrupt glutamate homeostasis,11 and reproduce NMO pathology in inflammatory autoimmune models in vivo.12,13 It still remains elusive, however, why other organs with high AQP4 expression such as the kidneys are not affected by the autoimmune process.

Different assays have been established to measure antibodies targeting AQP4 in NMO spectrum disease. While indirect immunofluorescence (IF) using mouse brain sections remains the gold standard for the detection of NMO-IgG,4 new quantitative IF, immunoprecipitation (IP),3,4,8,14,15 and radioimmunoprecipitation16 assays have been developed. Nevertheless, a significant proportion (about 40%-50%) of sera from patients with clinically definite NMO are negative for anti-AQP4 antibodies in the available assays. One possible explanation might be that none of the assays fully reflect the posttranslational modification of AQP4 that occurs in human glial cells, leading to a false-negative result. Given the potential functional importance of antibodies targeting conformational epitopes of membrane proteins in other CNS diseases and experimental models,1720 assays that precisely reflect expression of the protein in vivo are desperately needed.

Therefore, we established a bioassay that allows quantification of antibodies to human native AQP4 (nAQP4) expressed in a human astrocytoma cell line (nAQP4-IgG). The assay was validated with well-defined sets of sera and was used to investigate the biological activity of anti-nAQP4-IgG in vitro. Complement- and NK cell–mediated cytotoxic effects were observed with all sera containing antibodies to nAQP4. The strong correlation between antibody titer and in vitro cytotoxic effect suggests that the bioassay reliably detects biologically relevant antibodies in NMO and NMO spectrum diseases. Cell-based assays are powerful tools to investigate antibody responses to membrane proteins and may help identify and characterize antibody responses relevant in other autoimmune diseases.

PATIENTS AND CONTROLS

For initial validation of the assay, sera from 14 patients with NMO or opticospinal multiple sclerosis (MS) (13 women, 1 man) participating in clinical studies at the University of California, San Francisco were used. Patients with opticospinal MS were defined as those meeting International Panel Criteria for MS but having clinical attacks restricted to the optic nerves or spinal cord and disease duration of at least 5 years. All subjects provided written informed consent, and the studies received approval from the committee on human research.

For further evaluation of the assay, an independent cohort of 57 patients with NMO spectrum diseases was recruited from various neurology centers in Hungary. Sera and detailed clinical data were collected at the Department of Neurology, University of Pecs, Pecs, Hungary, and all patients were examined by neurologists specialized in neuroimmunology or MS care. Each patient has had brain and spinal cord magnetic resonance imaging (1.5 or 3 T). Based on clinical and paraclinical data, patients were diagnosed according to the revised criteria by Wingerchuk et al,21 with the exception of NMO-autoantibody status, which had not been determined beforehand. The cohort comprised 18 patients fulfilling the diagnostic criteria for NMO (15 women, 3 men; age range, 21-60 years; median age, 44 years), 12 patients with RION (all women; age range, 25-50 years; median age, 39 years), and 27 patients with LETM (20 women, 7 men; age range, 24-72 years; median age, 49 years).

This cohort was compared with 38 patients with MS (25 women, 13 men; age range, 18-66 years; median age, 41 years) fulfilling the revised McDonald criteria22 and 64 patients with other neurological diseases (ONDs) (16 women, 48 men; age range, 20-79 years; median age, 41 years) who were recruited in Germany. Patients with ONDs had human immunodeficiency virus infection, various extracerebral or intracerebral neoplasms, viral meningitis, headache, (pseudo)radicular pain, or vertigo.

CLONING AND EXPRESSION OF AQP4

The human M1 and M23 variants of AQP4 were expressed by a lentiviral transfection system in the LN18 cell line as described previously.17 The LN18 cell line is a human glioma cell line established from an astrocytoma biopsy specimen.23 Total human brain RNA (BD Biosciences, San Jose, California) was used to synthesize full-length complementary DNAs of the M1 and M23 AQP4 variants (with Spe I and Sac II restriction sites at 5′ and 3′ ends). The sequence of the common reverse primer used for both M1 and M23 variants was 5′-CCG CGG GTC TGC TTT CAG TGC GAT CTT CTA G-3′. Sequences of the forward primers were 5′-ACT AGT GCA ATG AGA GCT GCA CTC TGG CTG-3′ for the M1 variant and 5′-ACT AGT ATG GTG GCT TTC AAA GGG GTC TGG-3′ for the M23 variant. The polymerase chain reaction–amplified products were cloned into the plasmid pLenti6/V5 (Invitrogen Corp, Carlsbad, California) by using Spe I and Sac II restriction sites. To generate gene-containing virus particles, the pLenti6/V5-AQP4 M1 or M23 constructs and packaging mix were used to transfect a 293 FT cell line by Lipofectamine 2000 (Invitrogen Corp). After transfection of 48 hours, virus-containing supernatant was collected and used to transduce the human LN18 astrocytoma cell line to generate both M1- and M23-expressing cell lines (LN18AQP4 and LN18AQP4-M23, respectively). The LN18 cell line was also transduced with an empty vector pLenti6/V5 to establish a control cell line (LN18CTR). The 3 stably transduced cell lines were maintained under the same culture conditions and the same selection pressure throughout the experiments.

INDIRECT IF STAINING

The LN18AQP4 and LN18CTR cells were fixed for 10 minutes on ice with cytofix (BD Biosciences). After fixation, cells were blocked for 1 hour with blocking buffer containing 10% normal goat serum (Vector Laboratories, Inc, Burlingame, California). Extensive washing was performed at each step with washing buffer (0.05% Tween 20 [Sigma-Aldrich Co, St Louis, Missouri] in 1× phosphate-buffered saline [PBS]). After blocking, cells were incubated with polyclonal rabbit antirat AQP4 IgG antibody (Sigma-Aldrich Co), which cross-reacts with human AQP4. The antibody targets amino acids 249 to 323 of the intracellular domain of the rat protein, which is 95% similar to the human sequence. Cells were incubated with 200 μL of the antibody at a concentration of 1 μg/mL or with nAQP4-IgG–positive serum (diluted 1:1000 in blocking buffer). Cells were incubated for 2 hours on ice, washed 5 times with washing buffer, and incubated for 1 hour with Alexa Fluor-488–labeled goat antirabbit IgG or goat antihuman IgG (Molecular Probes; Invitrogen Corp). The nuclei of the cells were counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen Corp). Cells were mounted on slides in mounting medium (Vector Laboratories, Inc). Images were captured and analyzed with a fluorescence microscope (Fluorescence Microscope System, Carl Zeiss AG, Jena, Germany; or cell ˆR Imaging Station, Olympus, Essex, England).

WESTERN BLOT ANALYSIS

The LN18AQP4 and LN18CTR cells were lysed with RIPA buffer (Sigma-Aldrich Co). Five micrograms of total protein lysates of LN18AQP4 and LN18CTR was separated by using 10% Bis-Tris gels (Invitrogen Corp), and proteins were transferred to polyvinylidene fluoride membranes (Invitrogen Corp). The polyvinylidene fluoride membranes were blocked in blocking buffer (1× PBS containing 4% milk and 0.05% Tween 20) for 1 hour at room temperature. The membranes were probed with rabbit polyclonal anti-AQP4-IgG antibody (stock [1 mg/mL]) diluted in blocking buffer (diluted 1:2000). Membranes were incubated overnight at 4°C, washed 5 times with washing buffer (0.05% Tween 20 in 1× PBS), and incubated for 1 hour at room temperature with a horseradish peroxidase–conjugated goat antirabbit IgG (Fc) antibody (AbD Serotec, Raleigh, North Carolina) (1 mg/mL) diluted in blocking buffer (diluted 1:2000). Afterward, membranes were washed 5 times with washing buffer. Finally, antibody binding was detected by an Amersham ECL system (GE Healthcare, Piscataway, New Jersey) according to the manufacturer's instructions.

FLOW CYTOMETRY ANALYSIS

Flow cytometry was used first to confirm the expression of AQP4 in the LN18AQP4 cell lines and second to identify anti-AQP4 autoantibodies in patients' sera.

The expression of AQP4 in LN18AQP4 cells was determined by intracellular staining (for intracellular staining, cells were fixed and permeabilized with Cytofix and Cytoperm [1×] buffers [BD Biosciences], respectively) with rabbit anti-AQP4 polyclonal IgG antibody and Alexa Fluor-488–labeled goat antirabbit IgG antibody. Surface expression of AQP4 was determined by staining with an NMO-IgG–positive serum in combination with Alexa Fluor-488–labeled goat antihuman IgG antibody (Molecular Probes; Invitrogen Corp). The median fluorescence intensity (MFI) was used as the readout in all experiments.

We added 20 000 LN18AQP4 or LN18CTR cells in 20 μL of Roswell Park Memorial Institute (RPMI) 1640 growth medium to each well of 96-well plates containing 20 μL of 1:100 diluted primary antibody or NMO-IgG–positive serum. The plates were incubated on ice for 25 minutes on an orbital shaker. Cells were then washed twice with FACS buffer (1× PBS containing 1% fetal bovine serum). Alexa Fluor-488–labeled goat antirabbit IgG (Fc) or antihuman IgG (H + L) antibody (diluted 1:100 in FACS buffer) was added to each well and incubated on ice. After 25 minutes, cells were washed twice, resuspended in 175 μL of FACS buffer, and transferred into FACS tubes (BD Biosciences). Cells were analyzed with a CyAn ADP high-performance Flow Cytometer (Dako Corp, Glostrup, Denmark). To detect nAQP4-IgG antibodies in clinical samples, 20 000 LN18AQP4 and LN18CTR cells in 20 μL of RPMI medium were added to each well containing 20 μL of 1:100 diluted patient serum in duplicate and processed likewise. Screening was performed with Alexa Fluor-488–labeled goat antihuman IgG (H + L) as the secondary antibody. The IgG isotypes and IgM antibodies in positive samples were further determined by using secondary anti-IgG1–, anti-IgG2–, anti-IgG3–, and anti-IgG4–specific antibodies (Sigma-Aldrich Co) and anti-IgM–specific antibodies (Molecular Probes; Invitrogen Corp).

ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY ASSAY

We isolated CD56+ NK cells from peripheral blood mononuclear cells of healthy donors separated by density gradient centrifugation (Biocoll Separating Solution; Biochrom, Cambridge, England). We incubated 1 × 107 peripheral blood mononuclear cells with 20 μL of CD56 MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and 80 μL of MACS buffer (2.5 g of bovine serum albumin and 2 mL of 0.05M EDTA in 500 mL of 1× PBS) on ice on an orbital shaker for 15 minutes. After incubation, the cells were washed with and resuspended in 2 mL of MACS buffer. The CD56+ cells were then separated using magnetic cell separation (AutoMACS; Miltenyi Biotec). Purity of more than 95% NK cells was obtained.

Total IgG was purified from nAQP4-IgG–positive and nAQP4-IgG–negative sera by using Sepharose-G according to the manufacturer's protocol (GE Healthcare). We added 10 μg of purified IgG diluted in 40 μL of RPMI growth medium in duplicate to a U-shaped 96-well plate (CellStar; Greiner Bio-One, Monroe, North Carolina), each well containing 30 000 LN18CTR or LN18AQP4 cells in 40 μL of RPMI medium (final IgG concentration was 125 μg/mL) and incubated on ice for 25 minutes on an orbital shaker. Cells were then washed twice with FACS buffer (1% fetal calf serum in 1× PBS), washed once with RPMI medium, and transferred to a 96-well cell culture plate (F-bottom, CellStar). We added 60 000 CD56+ NK cells resuspended in 40 μL of RPMI medium to each well, and RPMI medium was adjusted to a final volume of 150 μL. After incubation at 37°C in a humidified carbon dioxide incubator for 12 hours, the supernatants were discarded. Remaining cells were detached by 70 μL of 0.05% trypsin-EDTA and transferred into FACS tubes (BD Biosciences). Cell number and viability were determined by flow cytometry (CyAn ADP) based on forward scatter and sideward scatter parameters.

COMPLEMENT-MEDIATED CYTOTOXICITY ASSAY

On a U-shaped 96-well plate (CellStar), each well was loaded with 50 000 LN18CTR or LN18AQP4 cells in 40 μL of RPMI medium. Total IgG from nAQP4-IgG–positive and nAQP4-IgG–negative sera was purified as described earlier, diluted in 40 μL of RPMI growth medium, and added to each well to give a final IgG concentration of 125 μg/mL, in duplicate. Cells were incubated on ice for 25 minutes on an orbital shaker, washed twice with FACS buffer (1% fetal calf serum in 1× PBS) and once with RPMI medium, and transferred to a 96-well cell culture plate (F-bottom, CellStar). A total of 7.5 μL of nAQP4-IgG–negative serum was added as a complement source in each well, and RPMI medium was adjusted to a final volume of 150 μL (final dilution of control serum was 1:20). After incubation at 37°C in a humidified carbon dioxide incubator for 12 hours, the supernatants were discarded and the remaining cells were detached by adding 70 μL of 0.05% trypsin-EDTA and transferred into FACS tubes. Cell number and viability were measured by flow cytometry (CyAn ADP) based on forward scatter and sideward scatter parameters.

RECOMBINANT EXPRESSION OF nAQP4 IN A HUMAN ASTROCYTOMA CELL LINE

To investigate antibody reactivity to nAQP4, we expressed the human M1 AQP4 gene in the human astrocytoma cell line LN18 (LN18AQP4) by a lentiviral expression system (expression and detection strategy displayed in Figure 1A). In parallel, the LN18 cell line was transduced with an empty vector to establish an appropriate control cell line (LN18CTR). Expression of AQP4 in LN18AQP4 but not LN18CTR was demonstrated by Western blot (Figure 1B) using a polyclonal rabbit anti-AQP4-IgG (Fc) antibody. A high level of AQP4 expression was confirmed by immunocytochemistry and flow cytometry (Figure 1C and D) on permeabilized LN18AQP4 cells.

Place holder to copy figure label and caption
Figure 1

Bioassay to determine antibodies to human native aquaporin 4 (AQP4). A, Strategy to establish the bioassay for the detection of antibodies binding to native AQP4. The glioma cell line LN18 was stably transduced with either the M1 isoform of the AQP4 gene (to establish the LN18AQP4 cell line) or an empty vector (to establish the LN18CTR control cell line) using a lentiviral expression system (the LN18AQP4-M23 cell line expressing the M23 variant of AQP4 was produced likewise). The different antibodies used in the experiments are displayed. bp indicates base pairs. Expression of AQP4 was confirmed by Western blot of LN18AQP4 and LN18CTR cell lysates (primary antibody: polyclonal rabbit anti-AQP4 antibody; secondary antibody: goat antirabbit IgG) (B), immunocytochemistry (C), and flow cytometry on permeabilized cells (primary antibody: polyclonal rabbit anti-AQP4 antibody; secondary antibody: goat antirabbit IgG) (D).

Graphic Jump Location

Second, we verified the cell surface expression of AQP4 on LN18AQP4 by incubating AQP4-transfected cells with an NMO-IgG–positive serum (diluted 1:100). Immunocytochemistry with a fluorescence-labeled antihuman IgG-specific secondary antibody revealed appropriate labeling of LN18AQP4 but not LN18CTR cells (Figure 2A). Binding of serum NMO-IgG could also be visualized by flow cytometry in LN18AQP4 but not LN18CTR cells, confirming the suitability of the detection system (Figure 2B, lower panel). Similar results were obtained with the LN18 cell line transfected with the M23 variant of AQP4 (LN18AQP4-M23, data not shown).

Place holder to copy figure label and caption
Figure 2

Detection of serum autoantibodies specific for native aquaporin 4. A, Immunofluorescence staining (original magnification ×10 [upper row] and ×63 [lower row]) was performed on LN18CTR (left) and LN18AQP4 (right) cells with serum from a neuromyelitis optica (NMO)–IgG–positive patient as the primary antibody source and Alexa Fluor-488–labeled goat antihuman IgG (Invitrogen Corp, Carlsbad, California) as the secondary antibody. B, Flow cytometry was performed with serum from a patient with multiple sclerosis (NMO-IgG negative [upper histogram]) and a patient with NMO (NMO-IgG positive [lower histogram]) on LN18AQP4 (green) and LN18CTR (red) cells. The difference in the median fluorescence intensity (MFI) obtained with the LN18AQP4 and LN18CTR cell lines is termed ΔMFI and corresponds to the concentration of aquaporin 4–specific serum antibodies. Sera from NMO-IgG–positive patients were tested at different concentrations for native aquaporin 4–specific antibodies in the bioassay. A representative example (C) and the dilution curves of 4 sera (D) are shown.

Graphic Jump Location
QUANTIFICATION OF SERUM ANTIBODIES TO nAQP4 BY A CELL-BASED BIOASSAY

Serum antibodies from healthy control donors did not bind to either of the 2 cell lines (Figure 2B, upper panel). Serial dilution of sera from NMO-IgG–positive patients demonstrated that the antibody to nAQP4 can be detected at a dilution of up to 1:100 000 (Figure 2C and D). After logarithmic transformation, dose-response curves were approximately linear (Figure 2D). This indicates that the bioassay is able to detect nAQP4-IgG autoantibodies in serum samples in a quantitative manner over a wide range of concentrations.

COMPARATIVE ANALYSIS OF NMO-IgG AND ANTI-nAQP4-IgG

Next, we wished to validate our assay in comparison with an already established assay for the detection of NMO-IgG. Thus, our assay was applied to 14 serum samples from patients with clinically defined NMO or opticospinal MS that had been analyzed at the Department of Laboratory Medicine and Pathology, Mayo Clinic by IP and IF assays, the gold-standard approaches for the detection of NMO-IgG (Table). Eight samples tested positive by IF and IP, 1 sample tested positive by IP but not IF, and 5 samples were negative in both assays. In our assay, all 9 NMO-IgG–positive samples were strongly positive, although titers differed by more than 1 order of magnitude. Interestingly, the difference in the MFI (ΔMFI) of the IP-positive/IF-negative serum was in the midrange. Further, 4 of 5 NMO-IgG–negative sera were below the cutoff of our assay as well, but 1 NMO-IgG–negative serum tested strongly positive. Although these data are derived from a rather small sample set and are thus preliminary, the results suggest that our cell-based assay is comparable to the current gold-standard NMO-IgG assays in terms of sensitivity and specificity.

Table Graphic Jump LocationTable Comparison of Neuromyelitis Optica–IgG Reactivity and Native Aquaporin 4 Fluorescence Intensity in the Cell-Based Assay

Similar results were obtained with the LN18 cell line transfected with the M23 variant of AQP4 (LN18AQP4-M23), although serum antibody binding seemed to be slightly lower with this cell line compared with the LN18AQP4 cell line (data not shown).Therefore, all further analyses were performed with the LN18AQP4 cell line.

PREVALENCE OF ANTI-nAQP4-IgG IN NMO SPECTRUM DISEASES

To determine the sensitivity and specificity of our assay, we applied the assay to masked serum samples from patients with NMO, LETM, RION, MS, and OND (Figure 3A). Sera were diluted 1:100 and tested in duplicates using ΔMFI as compared with background staining as the readout.

Place holder to copy figure label and caption
Figure 3

Prevalence and isotype analysis of autoantibodies to native aquaporin 4 (nAQP4). A, Sera of different patients stratified according to their clinical diagnosis were analyzed for the prevalence of nAQP4-IgG. Each diamond represents 1 sample. The difference in median fluorescence intensity (ΔMFI) is shown for each group and the diagnostic cutoff is indicated by the dashed line. OND indicates other neurological disease; MS, multiple sclerosis; NMO, neuromyelitis optica; LETM, longitudinally extensive transverse myelitis; and RION, relapsing inflammatory optic neuritis. B, In 7 paired cerebrospinal fluid (CSF) and serum samples, anti-nAQP4-IgG antibodies were determined in parallel after adjusting for total IgG concentration. Corresponding CSF and serum ΔMFIs are plotted in relation to antibody indices (AIs) of 1.0 and 1.5. C, Analysis of IgG isotypes and IgM antibodies to nAQP4 is shown in 4 representative patients. D, Paired analysis of anti-nAQP4-IgG1 isotype levels are shown in relation to anti-nAQP4-IgG in positive samples.

Graphic Jump Location

To define a cutoff for anti-nAQP4-IgG antibody positivity, sera from 64 patients with ONDs were measured as negative controls. The ΔMFI ranged from −8.1 to 5.0, with a median of −0.63. The cutoff was thus set at a ΔMFI of 10, corresponding to twice the 99th percentile of the OND control group.

No sera from patients with MS tested positive for anti-nAQP4-IgG (median ΔMFI, −0.81; range, −8.3 to 5.1). In contrast, anti-nAQP4-IgG antibodies were present in 11 of 18 patients with clinical NMO (61.1%; median ΔMFI, 186; range, −3.5 to 1638.5), 5 of 12 patients with RION (41.7%; median ΔMFI, 1.8; range, −3.3 to 1414.0), and 10 of 27 patients with LETM (37.0%; median ΔMFI, 3.6; range, −6.7 to 1413.8). In 22 of 27 anti-nAQP4-IgG–positive patients, serum concentrations were considered high because they exceeded a ΔMFI of 100 (Figure 3A). Low titers were observed in 4 patients with LETM and 1 patient with NMO. The specificity and positive predictive value of our assay were 100%. The sensitivity was 57.9% with respect to NMO spectrum diseases; the negative predictive value was 66.6%. In line with previous findings,4,7 the highest number of patients with anti-nAQP4 antibodies and the highest titers were observed in the NMO group.

ISOTYPE OF ANTI-nAQP4-IgG

To determine the isotype of the anti-nAQP4-IgG, we analyzed all positive serum samples using secondary antibodies specific for human IgG1, IgG2, IgG3, and IgG4. Significant levels of nAQP4-specific antibodies were found only when the secondary antibody targeted IgG1 (Figure 3B). In line with this finding, a strong correlation between the levels of anti-nAQP4-IgG and anti-nAQP4-IgG1 was observed (Figure 3C). IgM antibodies to nAQP4 were not found in any of the anti-nAPQ4-IgG–positive patients at a serum dilution of 1:100.

DETECTION OF ANTI-nAQP4-IgG IN CEREBROSPINAL FLUID

To address the question of intrathecal production of anti-nAQP4-IgG antibodies, we determined the antibody index (AI). The AI is described by the formula AI = Qspec/QIgG, with Q representing the cerebrospinal fluid (CSF)/serum quotient of nAQP4-IgG antibodies (Qspec) or total IgG (QIgG). An AI greater than 1.5 indicates a production of antibodies in the CNS compartment by sequestered plasma cells or plasma blasts (intrathecal synthesis of the specific antibody).24 Corresponding CSF samples from anti-nAQP4-IgG–positive sera were available from 7 patients with NMO spectrum diseases in whom CSF and serum samples had been collected at the same time. After adjusting for total IgG concentration (QIgG = 1), pairs of serum and CSF samples were subjected to the nAQP4 assay in parallel. In 3 of 7 paired samples, the AI was between 1.0 and 1.5; in the remaining 4 paired samples, the AI was below 1.0 (Figure 3D). Thus, we have no evidence of intrathecal nAQP4-IgG synthesis in this preliminary analysis.

BIOLOGICAL ACTIVITY OF ANTI-nAQP4-IgG

To investigate whether nAQP4-IgG is biologically active, we tested the serum IgG antibodies for their ability to induce a cytotoxic effect in LN18AQP4 glioma cells in the presence of complement or human NK cells (Figure 4). In the presence of NK cells, IgG from anti-nAQP4-IgG–positive sera induced a specific, dose-dependent cytotoxic effect in LN18AQP4 cells but not LN18CTR cells (Figure 4A and B). This effect was evident even at an IgG concentration of 1.25 μg/mL (Figure 4A). The extent of NK cell–mediated cytotoxic effect strongly correlated with the serum concentration of anti-nAQP4-IgG (r = 0.8763; P = .01) (Figure 4C). We also observed a complement-mediated cytotoxic effect in LN18AQP4 cells incubated with IgG purified from anti-nAQP4-IgG–positive sera (Figure 4D). The extent of killing was less pronounced compared with the NK cell–mediated killing but again correlated well with the serum levels of anti-nAQP4-IgG (r = 0.9515; P = .001).

Place holder to copy figure label and caption
Figure 4

Cytotoxic activity of native aquaporin 4 (nAQP4)–IgG. A, The LN18AQP4 and LN18CTR cells were incubated with different concentrations of total IgG purified from a high-titer anti-nAQP4-IgG serum (125 μg/mL, 62.5 μg/mL, 25 μg/mL, and 1.25 μg/mL). Survival of LN18 cells in the presence of natural killer (NK) cells was determined after 12 hours. B, The LN18AQP4 and LN18CTR cells were incubated with 125-μg/mL total IgG purified from anti-nAQP4-IgG–positive sera of patients with neuromyelitis optica or anti-nAQP4-IgG–negative patients with multiple sclerosis (MS). Survival of LN18 cells in the presence of NK cells was determined after 12 hours and normalized to the negative control sample (LN18 cells alone). The experiment was performed in duplicate; mean and standard deviation are shown. C, Correlation of the antibody-dependent cell-mediated cytotoxic in vitro activity (cell killing of LN18AQP4 minus cell killing of LN18CTR in the presence of serum IgG and NK cells) with different anti-nAQP4-IgG concentrations. Each dot represents the analysis of 1 patient's serum. D, The LN18AQP4 and LN18CTR cells were incubated with 125-μg/mL total IgG purified from nAQP4-IgG–positive sera of patients with NMO or nAQP4-IgG–negative sera of patients. Survival of LN18 cells in the presence of anti-nAQP4-IgG–negative serum as a complement source was determined after 12 hours and normalized to the negative control sample (LN18 cells alone).

Graphic Jump Location

We present data of our novel bioassay for the detection of nAQP4-IgG. The advantage of our assay is 2-fold. First, the target antigen is expressed in a human cell line of glial origin and displayed on the membrane of the cells, similar to the expression in human astrocytes. Second, only those antibody reactivities that are directed against extracellular epitopes are measured. We demonstrate that these antibodies confer biological activity and may thus be pathogenetically relevant because we provide evidence for anti-AQP4-IgG antibody–mediated cytotoxic effect.

Aquaporin 4 is hypothesized to be the major autoimmune target in NMO spectrum diseases.4 It is a membrane protein with several transmembrane domains that exists in at least 2 isoforms.25,26 It is expressed in astrocytes but also in the distal collecting ducts of the kidney and parietal cells of the stomach.27 Antibodies to AQP4 are found in a significant proportion of patients with NMO, RION, and LETM but not in those with MS or other autoimmune diseases of the CNS.4,5,1416,27 Interestingly, autoimmunity seems to exclusively target AQP4 in the CNS as neither renal nor parietal cell dysfunction has been described in patients with NMO. This may suggest that the AQP4 protein expressed in the brain differs from the protein expressed outside the CNS. It is well known that tissue-specific posttranslational modifications that alter protein structure and function may occur. In the case of NMO spectrum diseases, expression of AQP4 in astrocytes may render the protein particularly immunogenic. Alternatively, astrocytes might be more vulnerable to anti-AQP4 antibody–mediated cytotoxic effect. Both conditions may account for the CNS-confined autoimmune reaction observed in NMO spectrum diseases.

To reflect the posttranslational modifications that might occur in astrocytes, we established a novel bioassay using a human AQP4-transfected cell line of glial origin. The glial cell line allowed us to express AQP4 protein at high levels on the surface of the cells, possibly with the posttranslational modifications occurring in astrocytes. This bioassay is at least as sensitive as the gold-standard assay, which was used to identify NMO-IgG.3,28 Its sensitivity and specificity also seem comparable to or even better than those of other assays that are based on transient transfection of AQP4 in cells of nonglial origin.6,29,30 Moreover, the flow-based assay allows high-throughput analysis and quantification of antibody titers to AQP4, which simplifies testing for this autoantibody.

The new assay allowed characterization of the isotype and biological activity of antibodies that target AQP4. We confirmed the high prevalence of anti-nAQP4-IgG autoantibodies in a cohort of patients with NMO spectrum disease who had been selected solely on the basis of clinical criteria. The antibody response to nAQP4 was almost exclusively IgG1. We found little evidence for a quantitative production of AQP4-specific antibodies in the CSF compartment, although locally produced antibodies may rapidly bind AQP4 in the CNS and thus escape detection by the assay. The AQP4-positive sera very efficiently induced complement- and NK cell–dependent cytotoxic effects of AQP4-expressing glioma cells. Interestingly, nAQP4-IgG concentrations correlated strongly with the extent of cytotoxic effect, suggesting that the antibodies detected with our assay represent the biologically active fraction of antibodies targeting AQP4 in vivo. It is possible that the serum anti-nAQP4-IgG concentration might be useful to monitor disease activity in NMO spectrum diseases.7,9 It is yet unclear why one-third of patients with NMO spectrum disease are AQP4 antibody negative in our assay. It is still possible that AQP4 may undergo posttranslational modifications and clustering, which are not achieved even in a transfected tumor cell line derived from astrocytes. Alternatively, these AQP4 antibody–negative patients with NMO may have another yet unknown autoantigen targeted in the CNS.

Besides the implications for the understanding of NMO spectrum diseases, our findings underscore the need for improved assay systems for detection of autoantibodies that target membrane proteins in human autoimmune diseases. The importance of antibodies targeting native vs denatured proteins was demonstrated in experimental autoimmune encephalomyelitis models induced by human myelin oligodendrocyte glycoprotein. While antibodies reactive to both denatured and native myelin oligodendrocyte glycoprotein are found in myelin oligodendrocyte glycoprotein–immunized animals, only antibodies targeting native myelin oligodendrocyte glycoprotein can mediate demyelination in vivo.31 Therefore, expression systems displaying the protein in the proper conformation are desperately needed to identify antibodies of relevance for human autoimmune diseases. Such bioassays not only allow more accurate antibody measurement but also enable determination of their biological activity with respect to various mechanisms of antibody-mediated cytotoxic effect. The implementation of such bioassays for the search for autoantibodies in human autoimmune diseases might allow us to discover and characterize antibodies that have escaped identification by screening with conventional assays.

Correspondence: Bernhard Hemmer, MD, Department of Neurology, Klinikum rechts der Isar, Technische Universität München, Ismaninger Strasse 22, 81675 München, Germany (hemmer@lrz.tu-muenchen.de).

Accepted for Publication: February 25, 2010.

Author Contributions:Study concept and design: Kalluri, Bennett, and Hemmer. Acquisition of data: Kalluri, Illes, Srivastava, Cree, Menge, and Berthele. Analysis and interpretation of data: Kalluri, Cree, Bennett, Berthele, and Hemmer. Drafting of the manuscript: Kalluri, Srivastava, Cree, Bennett, Berthele, and Hemmer. Critical revision of the manuscript for important intellectual content: Kalluri, Illes, Cree, Menge, Bennett, and Berthele. Statistical analysis: Hemmer. Obtained funding: Hemmer. Administrative, technical, and material support: Kalluri, Illes, Srivastava, Cree, Menge, Bennett, and Hemmer. Study supervision: Hemmer.

Financial Disclosure: None reported.

Funding/Support: This work was supported by grant He2386/7-1 from Deutsche Forschungsgemeinschaft and the Kompetenznetz Multiple Sklerose (Control-MS). Dr Bennett was supported by grant RG4320 from the National Multiple Sclerosis Society.

Wingerchuk  DMLennon  VALucchinetti  CFPittock  SJWeinshenker  BG The spectrum of neuromyelitis optica. Lancet Neurol 2007;6 (9) 805- 815
PubMed Link to Article
Jarius  SPaul  FFranciotta  D  et al.  Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat Clin Pract Neurol 2008;4 (4) 202- 214
PubMed
Lennon  VAWingerchuk  DMKryzer  TJ  et al.  A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364 (9451) 2106- 2112
PubMed Link to Article
Lennon  VAKryzer  TJPittock  SJVerkman  ASHinson  SR IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202 (4) 473- 477
PubMed Link to Article
Roemer  SFParisi  JELennon  VA  et al.  Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis. Brain 2007;130 (pt 5) 1194- 1205
PubMed Link to Article
Takahashi  TFujihara  KNakashima  I  et al.  Anti-aquaporin-4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain 2007;130 (pt 5) 1235- 1243
PubMed Link to Article
Jarius  SAboul-Enein  FWaters  P  et al.  Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain 2008;131 (pt 11) 3072- 3080
PubMed Link to Article
Hinson  SRPittock  SJLucchinetti  CF  et al.  Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica. Neurology 2007;69 (24) 2221- 2231
PubMed Link to Article
Hinson  SR McKeon  AFryer  JPApiwattanakul  MLennon  VAPittock  SJ Prediction of neuromyelitis optica attack severity by quantitation of complement-mediated injury to aquaporin-4–expressing cells. Arch Neurol 2009;66 (9) 1164- 1167
PubMed Link to Article
Vincent  TSaikali  PCayrol  R  et al.  Functional consequences of neuromyelitis optica-IgG astrocyte interactions on blood-brain barrier permeability and granulocyte recruitment. J Immunol 2008;181 (8) 5730- 5737
PubMed Link to Article
Hinson  SRRoemer  SFLucchinetti  CF  et al.  Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2. J Exp Med 2008;205 (11) 2473- 2481
PubMed Link to Article
Bennett  JLLam  CKalluri  SR  et al.  Intrathecal pathogenic anti-aquaporin-4 antibodies in early neuromyelitis optica. Ann Neurol 2009;66 (5) 617- 629
PubMed Link to Article
Bradl  MMisu  TTakahashi  T  et al.  Neuromyelitis optica: pathogenicity of patient immunoglobulin in vivo. Ann Neurol 2009;66 (5) 630- 643
PubMed Link to Article
Tanaka  MTanaka  KKomori  MSaida  T Anti-aquaporin 4 antibody in Japanese multiple sclerosis: the presence of optic spinal multiple sclerosis without long spinal cord lesions and anti-aquaporin 4 antibody. J Neurol Neurosurg Psychiatry 2007;78 (9) 990- 992
PubMed Link to Article
Matsuoka  TMatsushita  TKawano  Y  et al.  Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesions in multiple sclerosis in Japanese. Brain 2007;130 (pt 5) 1206- 1223
PubMed Link to Article
Paul  FJarius  SAktas  O  et al.  Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica. PLoS Med 2007;4 (4) e133
PubMed Link to Article
Zhou  DSrivastava  RNessler  S  et al.  Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis. Proc Natl Acad Sci U S A 2006;103 (50) 19057- 19062
PubMed Link to Article
Breithaupt  CSchubart  AZander  H  et al.  Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein. Proc Natl Acad Sci U S A 2003;100 (16) 9446- 9451
PubMed Link to Article
Iglesias  ABauer  JLitzenburger  TSchubart  ALinington  C T- and B-cell responses to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis and multiple sclerosis. Glia 2001;36 (2) 220- 234
PubMed Link to Article
O’Connor  KC McLaughlin  KADe Jager  PL  et al.  Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein. Nat Med 2007;13 (2) 211- 217
PubMed Link to Article
Wingerchuk  DMLennon  VAPittock  SJLucchinetti  CFWeinshenker  BG Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66 (10) 1485- 1489
PubMed Link to Article
Polman  CHReingold  SCEdan  G  et al.  Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria.” Ann Neurol 2005;58 (6) 840- 846
PubMed Link to Article
Diserens  ACde Tribolet  NMartin-Achard  AGaide  ACSchnegg  JFCarrel  S Characterization of an established human malignant glioma cell line: LN-18. Acta Neuropathol 1981;53 (1) 21- 28
PubMed Link to Article
Reiber  H Cerebrospinal fluid: physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases. Mult Scler 1998;4 (3) 99- 107
PubMed Link to Article
Engel  AFujiyoshi  YGonen  TWalz  T Junction-forming aquaporins. Curr Opin Struct Biol 2008;18 (2) 229- 235
PubMed Link to Article
Verkman  ASMitra  AK Structure and function of aquaporin water channels. Am J Physiol Renal Physiol 2000;278 (1) F13- F28
PubMed
Amiry-Moghaddam  MOttersen  OP The molecular basis of water transport in the brain. Nat Rev Neurosci 2003;4 (12) 991- 1001
PubMed Link to Article
McKeon  AFryer  JPApiwattanakul  M  et al.  Diagnosis of neuromyelitis spectrum disorders: comparative sensitivities and specificities of immunohistochemical and immunoprecipitation assays. Arch Neurol 2009;66 (9) 1134- 1138
PubMed Link to Article
Waters  PJarius  SLittleton  E  et al.  Aquaporin-4 antibodies in neuromyelitis optica and longitudinally extensive transverse myelitis. Arch Neurol 2008;65 (7) 913- 919
PubMed Link to Article
Fazio  RMalosio  MLLampasona  V  et al.  Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler 2009;15 (10) 1153- 1163
PubMed Link to Article
von Büdingen  HCHauser  SLOuallet  JCTanuma  NMenge  TGenain  CP Frontline: epitope recognition on the myelin/oligodendrocyte glycoprotein differentially influences disease phenotype and antibody effector functions in autoimmune demyelination. Eur J Immunol 2004;34 (8) 2072- 2083
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1

Bioassay to determine antibodies to human native aquaporin 4 (AQP4). A, Strategy to establish the bioassay for the detection of antibodies binding to native AQP4. The glioma cell line LN18 was stably transduced with either the M1 isoform of the AQP4 gene (to establish the LN18AQP4 cell line) or an empty vector (to establish the LN18CTR control cell line) using a lentiviral expression system (the LN18AQP4-M23 cell line expressing the M23 variant of AQP4 was produced likewise). The different antibodies used in the experiments are displayed. bp indicates base pairs. Expression of AQP4 was confirmed by Western blot of LN18AQP4 and LN18CTR cell lysates (primary antibody: polyclonal rabbit anti-AQP4 antibody; secondary antibody: goat antirabbit IgG) (B), immunocytochemistry (C), and flow cytometry on permeabilized cells (primary antibody: polyclonal rabbit anti-AQP4 antibody; secondary antibody: goat antirabbit IgG) (D).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2

Detection of serum autoantibodies specific for native aquaporin 4. A, Immunofluorescence staining (original magnification ×10 [upper row] and ×63 [lower row]) was performed on LN18CTR (left) and LN18AQP4 (right) cells with serum from a neuromyelitis optica (NMO)–IgG–positive patient as the primary antibody source and Alexa Fluor-488–labeled goat antihuman IgG (Invitrogen Corp, Carlsbad, California) as the secondary antibody. B, Flow cytometry was performed with serum from a patient with multiple sclerosis (NMO-IgG negative [upper histogram]) and a patient with NMO (NMO-IgG positive [lower histogram]) on LN18AQP4 (green) and LN18CTR (red) cells. The difference in the median fluorescence intensity (MFI) obtained with the LN18AQP4 and LN18CTR cell lines is termed ΔMFI and corresponds to the concentration of aquaporin 4–specific serum antibodies. Sera from NMO-IgG–positive patients were tested at different concentrations for native aquaporin 4–specific antibodies in the bioassay. A representative example (C) and the dilution curves of 4 sera (D) are shown.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3

Prevalence and isotype analysis of autoantibodies to native aquaporin 4 (nAQP4). A, Sera of different patients stratified according to their clinical diagnosis were analyzed for the prevalence of nAQP4-IgG. Each diamond represents 1 sample. The difference in median fluorescence intensity (ΔMFI) is shown for each group and the diagnostic cutoff is indicated by the dashed line. OND indicates other neurological disease; MS, multiple sclerosis; NMO, neuromyelitis optica; LETM, longitudinally extensive transverse myelitis; and RION, relapsing inflammatory optic neuritis. B, In 7 paired cerebrospinal fluid (CSF) and serum samples, anti-nAQP4-IgG antibodies were determined in parallel after adjusting for total IgG concentration. Corresponding CSF and serum ΔMFIs are plotted in relation to antibody indices (AIs) of 1.0 and 1.5. C, Analysis of IgG isotypes and IgM antibodies to nAQP4 is shown in 4 representative patients. D, Paired analysis of anti-nAQP4-IgG1 isotype levels are shown in relation to anti-nAQP4-IgG in positive samples.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4

Cytotoxic activity of native aquaporin 4 (nAQP4)–IgG. A, The LN18AQP4 and LN18CTR cells were incubated with different concentrations of total IgG purified from a high-titer anti-nAQP4-IgG serum (125 μg/mL, 62.5 μg/mL, 25 μg/mL, and 1.25 μg/mL). Survival of LN18 cells in the presence of natural killer (NK) cells was determined after 12 hours. B, The LN18AQP4 and LN18CTR cells were incubated with 125-μg/mL total IgG purified from anti-nAQP4-IgG–positive sera of patients with neuromyelitis optica or anti-nAQP4-IgG–negative patients with multiple sclerosis (MS). Survival of LN18 cells in the presence of NK cells was determined after 12 hours and normalized to the negative control sample (LN18 cells alone). The experiment was performed in duplicate; mean and standard deviation are shown. C, Correlation of the antibody-dependent cell-mediated cytotoxic in vitro activity (cell killing of LN18AQP4 minus cell killing of LN18CTR in the presence of serum IgG and NK cells) with different anti-nAQP4-IgG concentrations. Each dot represents the analysis of 1 patient's serum. D, The LN18AQP4 and LN18CTR cells were incubated with 125-μg/mL total IgG purified from nAQP4-IgG–positive sera of patients with NMO or nAQP4-IgG–negative sera of patients. Survival of LN18 cells in the presence of anti-nAQP4-IgG–negative serum as a complement source was determined after 12 hours and normalized to the negative control sample (LN18 cells alone).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable Comparison of Neuromyelitis Optica–IgG Reactivity and Native Aquaporin 4 Fluorescence Intensity in the Cell-Based Assay

References

Wingerchuk  DMLennon  VALucchinetti  CFPittock  SJWeinshenker  BG The spectrum of neuromyelitis optica. Lancet Neurol 2007;6 (9) 805- 815
PubMed Link to Article
Jarius  SPaul  FFranciotta  D  et al.  Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat Clin Pract Neurol 2008;4 (4) 202- 214
PubMed
Lennon  VAWingerchuk  DMKryzer  TJ  et al.  A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364 (9451) 2106- 2112
PubMed Link to Article
Lennon  VAKryzer  TJPittock  SJVerkman  ASHinson  SR IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202 (4) 473- 477
PubMed Link to Article
Roemer  SFParisi  JELennon  VA  et al.  Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis. Brain 2007;130 (pt 5) 1194- 1205
PubMed Link to Article
Takahashi  TFujihara  KNakashima  I  et al.  Anti-aquaporin-4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain 2007;130 (pt 5) 1235- 1243
PubMed Link to Article
Jarius  SAboul-Enein  FWaters  P  et al.  Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain 2008;131 (pt 11) 3072- 3080
PubMed Link to Article
Hinson  SRPittock  SJLucchinetti  CF  et al.  Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica. Neurology 2007;69 (24) 2221- 2231
PubMed Link to Article
Hinson  SR McKeon  AFryer  JPApiwattanakul  MLennon  VAPittock  SJ Prediction of neuromyelitis optica attack severity by quantitation of complement-mediated injury to aquaporin-4–expressing cells. Arch Neurol 2009;66 (9) 1164- 1167
PubMed Link to Article
Vincent  TSaikali  PCayrol  R  et al.  Functional consequences of neuromyelitis optica-IgG astrocyte interactions on blood-brain barrier permeability and granulocyte recruitment. J Immunol 2008;181 (8) 5730- 5737
PubMed Link to Article
Hinson  SRRoemer  SFLucchinetti  CF  et al.  Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2. J Exp Med 2008;205 (11) 2473- 2481
PubMed Link to Article
Bennett  JLLam  CKalluri  SR  et al.  Intrathecal pathogenic anti-aquaporin-4 antibodies in early neuromyelitis optica. Ann Neurol 2009;66 (5) 617- 629
PubMed Link to Article
Bradl  MMisu  TTakahashi  T  et al.  Neuromyelitis optica: pathogenicity of patient immunoglobulin in vivo. Ann Neurol 2009;66 (5) 630- 643
PubMed Link to Article
Tanaka  MTanaka  KKomori  MSaida  T Anti-aquaporin 4 antibody in Japanese multiple sclerosis: the presence of optic spinal multiple sclerosis without long spinal cord lesions and anti-aquaporin 4 antibody. J Neurol Neurosurg Psychiatry 2007;78 (9) 990- 992
PubMed Link to Article
Matsuoka  TMatsushita  TKawano  Y  et al.  Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesions in multiple sclerosis in Japanese. Brain 2007;130 (pt 5) 1206- 1223
PubMed Link to Article
Paul  FJarius  SAktas  O  et al.  Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica. PLoS Med 2007;4 (4) e133
PubMed Link to Article
Zhou  DSrivastava  RNessler  S  et al.  Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis. Proc Natl Acad Sci U S A 2006;103 (50) 19057- 19062
PubMed Link to Article
Breithaupt  CSchubart  AZander  H  et al.  Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein. Proc Natl Acad Sci U S A 2003;100 (16) 9446- 9451
PubMed Link to Article
Iglesias  ABauer  JLitzenburger  TSchubart  ALinington  C T- and B-cell responses to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis and multiple sclerosis. Glia 2001;36 (2) 220- 234
PubMed Link to Article
O’Connor  KC McLaughlin  KADe Jager  PL  et al.  Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein. Nat Med 2007;13 (2) 211- 217
PubMed Link to Article
Wingerchuk  DMLennon  VAPittock  SJLucchinetti  CFWeinshenker  BG Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66 (10) 1485- 1489
PubMed Link to Article
Polman  CHReingold  SCEdan  G  et al.  Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria.” Ann Neurol 2005;58 (6) 840- 846
PubMed Link to Article
Diserens  ACde Tribolet  NMartin-Achard  AGaide  ACSchnegg  JFCarrel  S Characterization of an established human malignant glioma cell line: LN-18. Acta Neuropathol 1981;53 (1) 21- 28
PubMed Link to Article
Reiber  H Cerebrospinal fluid: physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases. Mult Scler 1998;4 (3) 99- 107
PubMed Link to Article
Engel  AFujiyoshi  YGonen  TWalz  T Junction-forming aquaporins. Curr Opin Struct Biol 2008;18 (2) 229- 235
PubMed Link to Article
Verkman  ASMitra  AK Structure and function of aquaporin water channels. Am J Physiol Renal Physiol 2000;278 (1) F13- F28
PubMed
Amiry-Moghaddam  MOttersen  OP The molecular basis of water transport in the brain. Nat Rev Neurosci 2003;4 (12) 991- 1001
PubMed Link to Article
McKeon  AFryer  JPApiwattanakul  M  et al.  Diagnosis of neuromyelitis spectrum disorders: comparative sensitivities and specificities of immunohistochemical and immunoprecipitation assays. Arch Neurol 2009;66 (9) 1134- 1138
PubMed Link to Article
Waters  PJarius  SLittleton  E  et al.  Aquaporin-4 antibodies in neuromyelitis optica and longitudinally extensive transverse myelitis. Arch Neurol 2008;65 (7) 913- 919
PubMed Link to Article
Fazio  RMalosio  MLLampasona  V  et al.  Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler 2009;15 (10) 1153- 1163
PubMed Link to Article
von Büdingen  HCHauser  SLOuallet  JCTanuma  NMenge  TGenain  CP Frontline: epitope recognition on the myelin/oligodendrocyte glycoprotein differentially influences disease phenotype and antibody effector functions in autoimmune demyelination. Eur J Immunol 2004;34 (8) 2072- 2083
PubMed Link to Article

Correspondence

CME
Also Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
Please click the checkbox indicating that you have read the full article in order to submit your answers.
Your answers have been saved for later.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Web of Science® Times Cited: 41

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections
PubMed Articles