Table 1.
Basic clinical information of the cohort study.
Fig 1.
Reactivity of serum and CSF samples with HEK293 cells expressing GAD67.
Fixed HEK293 cells transfected to express GAD67 were incubated with serum and CSF of patient A and B (in green) and a commercial antibody against GAD67 (in red). The nuclei of the cells are stained with 4′,6-diamidino-2-phenylindole (DAPI). The merged reactivity is shown in panels C, F, I, and L. The CSF, but not the serum, of patient A immunoreacted against GAD67, whereas the serum and CSF of patient B were both positive. Scale bar = 20μm.
Fig 2.
GAD65 antibody levels measured by ELISA in serum (A) and CSF (B) samples classified by neurological syndrome.
CSF, but not serum, GAD65 antibody titres were significantly higher in the groups of ataxia and limbic encephalitis compared to those of stiff-person syndrome (median: 2.1 x103 U/ml (interquartile range (IQR):1.4–8.5) in SPS vs. 10.4 x103 U/ml (4.1–21.9) in CA; **p = 0.01 and 12 x103 U/ml (8.5–31.9) in LE; *p = 0.02). SPS: stiff person syndrome; CA: cerebellar ataxia; LE: limbic encephalitis; EP: epilepsy.
Fig 3.
GAD65 antibody titres measured by ELISA in serum samples from patients with (n = 93) or without (n = 13) additional GAD67 antibodies.
GAD65 antibody levels were higher in the group of patients with concurrent GAD67 antibodies. *p≤0.05.
Fig 4.
Immunoblot of purified GAD65 (upper panel) or GAD67 protein (lower panel).
Strips were incubated with a commercial antibody (A), serum from a healthy individual (B), and serum from patients with cerebellar ataxia (C and D), stiff-person syndrome (E), limbic encephalitis (F and G) and epilepsy (H and I). All patients’ sera reacted against GAD65 but only a few recognized GAD67 despite that all immunoreacted with HEK293 cells transfected with GAD67; this finding suggests that the recognized epitope is conformational. Both gels were run at the same time and blots were developed in parallel. The images were cropped to include the visible bands.
Fig 5.
Study of GAD65 epitope immunoreactivity.
Fixed HEK293 cells transfected to express full length GAD65, C-terminal (Ct), middle (PLP) and N-terminal (Nt) domains (all expressing an N-terminal myc tag) were incubated with patient serum (upper panels) or CSF (lower panels) (green) and a commercial antibody against myc tag (red). The nuclei of the cells are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The merged reactivity is shown in yellow. The serum sample reacts against full length and PLP domain, whereas the CSF sample shows a broader reactivity, staining the full length and three GAD65 domains. Scale bar = 20μm.
Fig 6.
Study of anti-GAD IgG internalization in cultures of dissociated rat hippocampal neurons that express GAD65.
Panel A demonstrates that neurons express GAD65; in this experiment neurons were permeabilized and incubated with a patient’s CSF with GAD65-ab (green) and a commercial GAD65-ab (red); the co-localization of reactivity with GAD65 is shown in yellow. Panels B and C show the experiment of internalization; in B, live neurons were incubated with CSF of a patient with anti-NMDAR antibodies, and in C with the CSF of a patient with GAD65 antibodies for 24h at room temperature. After blocking the extracellular IgG binding with a secondary antibody without fluorescent tag, the neurons were washed, fixed and permeabilized and the internalized IgG was determined with a secondary anti-human IgG antibody with a fluorescence tag (green), or with an antibody against MAP2 (red). Insets show dendrites at higher magnification. Only the CSF of the patient with IgG antibodies against NMDAR showed IgG internalization, as previously reported (Hughes et al. 2010), and used here as a control. Scale bar = 10μm, insert scale bar = 2,5μm.