Article Figures & Data
Figures
- Figure 1 ELISA Screen and Confirmatory TBA for Plexin D1-IgG
(A) Indirect ELISA for plexin D1-IgG in a previous cohort of 8 patients with NeP with plexin D1-IgG and 50 non-NeP patients (30 disease controls and 20 HCs) previously determined by TBA. Disease controls included 6 with amyotrophic lateral sclerosis; 4 each with multiple system atrophy, systemic lupus erythematosus, and neuro-Behçet disease; 3 with hereditary spinocerebellar degeneration; 2 each with Parkinson disease, normal pressure hydrocephalus, and Sjögren syndrome; and 1 each with Alzheimer disease, dementia with Lewy bodies, and corticobasal degeneration. The difference in OD values between plexin D1-coated wells and D1-uncoated wells (corrected OD value) was calculated, and the test was considered “ELISA-positive” when the corrected OD value was above the mean + 5 SD of the 50 plexin D1-IgG-negative controls determined by TBA (0.163, dotted line). Six of 8 patients with NeP with plexin D1-IgG by TBA were positive for plexin D1-IgG by ELISA, whereas all 50 disease controls and HCs without plexin D1-IgG by TBA were negative for plexin D1-IgG by ELISA. (B) Comparison of the newly established ELISA with TBA for plexin D1-IgG. The overall coincidence rate of ELISA to TBA was 96.6% (56/58). (C) Indirect ELISA for plexin D1-IgG in the present SFN cohort. The difference in OD values between plexin D1-coated wells and D1-uncoated wells (corrected OD value) was calculated, and the test was considered “ELISA-positive” when the corrected OD value was above 0.163 (dotted line). Plexin D1-IgG was positive in 8 of 63 (12.7%) of all patients with SFN, including 6 of 38 (15.8%) patients with iSFN, 2 of 25 (8.0%) patients with sSFN, and 2 of 55 (3.6%) HCs by ELISA. (D) IgG (green) from a representative patient with SFN (iSFN Case 1 in table 2) showed positive immunostaining of mouse small DRG neurons, whereas there was no significant immunoreactivity in 2 ELISA-seropositive HCs (HC 1 and HC 2). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (E) Correlation between the corrected OD value and the disease duration in patients with SFN with plexin D1-IgG (n = 8). There was a significant positive correlation between them (Spearman rank correlation; rs = 0.9639, p = 0.0001). Even after 1 outlier with the highest optical density was removed, the correlation between the corrected OD value and the disease duration remained significant (rs = 0.982, p < 0.0001). HC = healthy control; IgG = immunoglobulin G; iSFN = idiopathic small fiber neuropathy; OD = optical density; SFN = small fiber neuropathy; sSFN = secondary small fiber neuropathy; TBA = tissue-based indirect immunofluorescence assay.
- Figure 2 Assessment of Pain-Like Behaviors and Neuronal Activation in Passive Transfer Mice 24 Hours After Injection
(A) Mechanical pain hypersensitivity was assessed by calibrated von Frey filaments (0.04, 0.07, 0.16, 0.40, and 0.60 g) 24 hours after injection. Mice treated with purified IgG from patients 1, 2, and 3 showed significantly higher reaction rates to each stimulation strength (0.07, 0.16, 0.40, and 0.60 g filaments) than HC IgG-treated mice (1-way ANOVA with Dunnett test; *p < 0.01). No significant differences in reaction rates were seen with IgG from 2 inflammatory disease controls (1 patient with NBD and 1 patient with NPSLE) and patients 1, 2, and 3 after preadsorption with rhPlexin D1 compared with control IgG (1-way ANOVA with Dunnett test). (B) Indirect IFA of mouse L5 DRG sections revealed that IgG (green) from the patient with SFN (patient 3) with plexin D1-IgG but not control IgG bound to small DRG neurons. IgG from patient 3 preabsorbed with rhPlexin D1 (2 µg/mL) showed no significant immunoreactivity to mouse DRG. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). Immunostaining of pERK, a marker of primary afferent neuron activation, in L5 DRG of mice treated with purified IgG from patient 3 and control 24 hours after injection. Most of the pERK-labeled neurons in mice treated with purified IgG from patient 3 are small DRG neurons (≤25 µm in diameter). Few neurons are labeled for pERK in mice treated with control IgG and IgG from patient 3 after preabsorption with rhPlexin D1. Sections were counterstained with hematoxylin. (C) Distribution of pERK immunoreactivity among different-sized neurons was analyzed. DRG neurons were stratified as small (≤25 mm in diameter) and large (>25 mm in diameter) neurons. The pERK-labeled neurons in L5 DRG of mice treated with purified IgG from patients 1, 2, and 3 were predominantly small neurons of less than 25 μm in diameter. Scale bars = 50 µm and (inset) = 25 µm. ANOVA = analysis of variance; DRG = dorsal root ganglia; HC = healthy control; IFA = immunofluorescence assay; IgG = immunoglobulin G; NBD = neuro-Behçet disease; NPSLE = neuropsychiatric systemic lupus erythematosus; pERK = phosphorylated extracellular signal-regulated protein kinase; Pre-Ab = preabsorbed; rhPlexin D1 = recombinant human plexin D1.
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