PMC7732549_01
Clinical features (A) RNA immunoprecipitation assay to detect anti-signal recognition particle (SRP) antibodies. Serum and tonsil tissue from a patient with chronic tonsillitis was used as control. Each tonsil was ultrasonicated and lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate (SDS)] and a protease inhibitor cocktail (Complete mini EDTA-free, Roche) at a concentration of 40 mug/ml. Anti-SRP antibodies were detected using an RNA immunoprecipitation assay with extracts of HeLa cells as previously described. Briefly, 10 mul of serum or tonsil lysate was mixed with 2 mg of protein A-Sepharose CL-4B (Pharmacia Biotech AB) in 500 mul of immunoprecipitation buffer and incubated for 2 h. After washing three times with immunoprecipitation buffer, antigen-bound Sepharose beads were mixed with 100 mul of HeLa cell extract (6 x 10 cells equivalent per sample) for 2 h. Next, 30 mul of 3 M sodium acetate, 30 mul of 10% SDS, and 300 mul of phenol:chloroform:isoamyl alcohol (50:50:1, containing 0.1% 8-hydroxyquinoline) were added to extract the bound RNA. After ethanol precipitation, the RNA was resolved using a 7 M urea-8% polyacrylamide gel. The gel was silver-stained (Bio-Rad). Immunoprecipitated RNA located in the 7S RNA lesion (arrow) was regarded as anti-SRP antibody. Lane 1, total RNA; lane 2, patient's serum; lane 3, patient's tonsil lysate; lane 4, control serum; lane 5, control tonsil lysate; lane 6, positive control; lane 7, negative control; lane 8, empty. (B) Axial T1-weighted femoral MRI on admission showing severe femoral muscle atrophy with fat replacement. (C-E) Muscle biopsy sections of the left biceps. (C) Hematoxylin and eosin staining showed necrotizing myopathy with active necrosis and regeneration of muscle fibers with no lymphocyte infiltration. Scale bar, 100 mum. (D) Human leukocyte antigen-ABC staining indicated positive immunoreactivity in non-necrotic muscle fibers. Scale bar, 100 mum. (E) C5b-9 membrane attack complex staining exhibited immunopositive deposition on the surface of muscle fibers. Scale bar, 20 mum. (F) Axial T1-weighted femoral MRI on discharge showing recovery of muscular volume compared to (B).
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RNA immunoprecipitation revealed seropositivity for anti-SRP antibodies ( Figure 1A ). MRI detected limb muscle atrophy, especially in the triceps, quadriceps, hamstrings, and adductor magnus ( Figure 1B ). Muscle biopsy from the left biceps demonstrated myopathic changes with active myofiber necrosis and regeneration of muscle fibers without obvious lymphocyte infiltration ( Figure 1C ), mild expression of human leukocyte antigen-ABC in scattered non-regenerating fibers ( Figure 1D ), and deposition of C5b-9 membrane attack complex in the sarcolemma ( Figure 1E ). Serum anti-SRP antibody titers became undetectable after tonsillectomy ( Figure 3 ) although RNA immunoprecipitation did not detect anti-SRP antibodies in the resected tonsils ( Figure 1A ). Muscle weakness dramatically responded to IVIG, which was accompanied by a rapid increase in DeltaIgG ( Figure 3 ) and recovery of muscle volume ( Figure 1F ) and strength (grade 4-5/5 on the MRC scale with a sum score of 56 points) ( Figure 3 ).
Micrographs of resected tonsil sections Hematoxylin and eosin (H&E) staining showed chronic tonsillitis with activated macrophages based on CD68 positivity. Immunostaining for CD4 and CD8 displayed T cell accumulation in the opposite sides of blind crypts. Most lymphoid follicles (CD20, a marker for follicular B cell) consisted of primary follicles with poorly developed germinal centers (CD10, a marker for germinal center B cells; CD21, a marker for germinal center dendritic cells), rare secondary follicle formation, and an expanded mantle zone (IgD, a marker for mantle zone B cells), which are characteristic pathological features of chronic tonsillitis associated with IgA nephritis but not chronic tonsillitis without IgA nephritis. Scale bar, 1 mm.
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As shown in  Figure 2 , immunohistochemical analyses of resected tonsil sections showed chronic tonsillitis associated with macrophage activation based on hematoxylin and eosin (H&E) staining and CD68 (a marker of activated macrophages).
Clinical course and treatments during hospitalization. Tonsillectomy (red dotted line) dramatically improved the therapeutic response to IVIG and was accompanied by a rapid increase in DeltaIgG (increase in serum IgG levels 2 weeks after the start of IVIG treatment relative to pre-treatment levels). Purple square, Medical Research Council (MRC) sum score; light blue triangle, eGFR; magenta circle, anti-SRP antibody titer; green triangle, serum CK concentration; blue circle, %VC; orange diamond, DeltaIgG; IVMP, intravenous methylprednisolone pulse therapy; IVIG, intravenous immunoglobulin therapy.
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The first course of IVIG quickly normalized serum CK levels and partially improved %VC (82.8%), but limb muscle weakness and atrophy remained severe, with an MRC sum score of 46 points ( Figure 3 ). Multiple rounds of IVIG gradually increased %VC to 93.6%, but severe limb muscle weakness and atrophy persisted, with an MRC sum score of 48 points, and DeltaIgG remained low ( Figure 3 ), indicating persistent inflammation. Serum anti-SRP antibody titers became undetectable after tonsillectomy ( Figure 3 ) although RNA immunoprecipitation did not detect anti-SRP antibodies in the resected tonsils ( Figure 1A ). Muscle weakness dramatically responded to IVIG, which was accompanied by a rapid increase in DeltaIgG ( Figure 3 ) and recovery of muscle volume ( Figure 1F ) and strength (grade 4-5/5 on the MRC scale with a sum score of 56 points) ( Figure 3 ). In contrast, tonsillectomy did not improve IgA nephropathy-induced CKD status ( Figure 3 ).