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. 2022 Apr 11;13:854445. doi: 10.3389/fimmu.2022.854445

Figure 3.

Figure 3

Identification of the autoantibody in a patient with acute encephalitis. P1 patient was diagnosed with encephalitis. The CSF sample from a control patient with carpal tunnel syndrome (nCSF) and the serum from a healthy person (nSerum) was used as negative controls. (A) Immunostaining on rat brain sections. The CSF from P1 patient had stained on hippocampus and cerebellum. (B) P1 patient’s serum stained obviously in the cytosolic compartments of the neurons in the cortex, CA3 area of the hippocampus, and cerebellum on mouse brain sections (green). White arrows, possible oligodendrocytes; arrow heads, Purkinje cells. CRMP2-expressing HEK293T cell absorbed P1 patient’s serum had no obvious staining. Cell nuclei were stained blue with DAPI. (C) P1 patient’s serum (green) stained positively in the soma and dendrites of cultured mouse cortical neurons which were labeled with anti-MAP2 Ab (red). (D, E) IP of P1 CSF with rat brain protein lysate. In total, 500 μl (lane 1) and 100 μl (lane 2) P1 CSF were applied to IP with 10 mg total protein of fresh rat brain lysate, respectively. Moreover, 500 μl nCSF was used for negative control. A positive band around 70 kDa was pulled down by P1 CSF but not by nCSF. A similar protein size band (red arrowhead) was observed on a parallel gel with Coomassie brilliant blue staining (E). (F) Characterization of the autoantigen by mass spectrometry. The suspected protein band in (E) was collected for protein identification by LC-MS/MS. Seventeen identified peptides (red) matched rat DPYL2 (UniProtKB ID: P47942), an alias of CRMP2, with a score of 226 and sequence coverage of 40%. CRMP2, collapsin response mediator protein 2; CSF, cerebrospinal fluid; DAPI, 6-diamidino-2-phenylindole; DPYL2, dihydropyrimidinase like 2; GL, granular layer; IB, immunoblotting; IHC, immunohistochemistry; IP, immunoprecipitation; LC-MS/MS, liquid chromatography tandem mass spectrometry; MAP2, microtubule-associated protein 2; ML, molecular layer; PCL, Purkinje cell layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars represent 50 μm in (B) and 20 μm in (C).