Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described

β2-m amyloid fibrils are endocytosed into endosomes/lysosomes, leading to the disruption of their membranes.

<p>Representative electron micrographs of HIG-82 cells taken as described in Materials and Methods. HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer for 6 hrs (A), or 100 μg/ml β2-m fibrils for 2 hrs (B, C) or 6 hrs (D-F) as described in Materials and Methods. The inset in (B) is a higher magnification of the box. (B, D) HIG-82 cells were covered with amyloid fibrils. Note that a part of the plasma membrane invaginated and fused to form an endocytic vesicle containing amyloid fibrils (inset in B). (C, E) Many endosomes/lysosomes were filled with amyloid fibrils, and some endosomal/lysosomal membranes were disrupted by intravesicular fibrils. (F) Nuclear deformation, shrinkage, and chromatin condensation at the nuclear rim were also observed. The scale bars are 5 μm long in A, B, D and F and 1 μm long in C and E.</p

β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.

<p>After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.</p

Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.

<p>Representative electron micrographs of HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils for 6 hrs as described in Materials and Methods. Images were taken as described in Materials and Methods. (D-F) Higher magnifications of the boxes in A-C, respectively. Note that the endocytosed amyloid fibrils leaked from endosomal/lysosomal vesicles into the cytosol (A, D), and some fibrils were found adjacent to mitochondria (B, C, E, F). The scale bars are 500 nm long in A-C and 200 nm long in D-F.</p

β2-m amyloid fibrils are internalized and sorted to lysosomes.

<p>HIG-82 cells incubated with Ham’s F12 medium containing vehicle buffer, 10 μg/ml β2-m monomer, or 10 μg/ml β2-m fibrils for 12 hrs were stained for lysosomes (red), β2-m (green), and nuclei (blue), and observed with the confocal laser microscope as described in Materials and Methods. When the cells were incubated with fibrils (right column), green fluorescence indicating β2-m fibrils were observed inside the cells in a granular pattern, as well as on the surface of the cells. Importantly, some green-colored granules containing β2-m fibrils were merged with red-colored lysosomes. The scale bars are 10 μm long.</p

β2-m amyloid fibrils have no effect on artificial plasma membranes.

<p>(A) A representative light micrograph of large unilamellar vesicles (LUVs) containing carboxyfluorescein prepared as described in Materials and Methods. They were less than 50 μm in diameter. The scale bars are 50 μm long. After LUVs were incubated with β2-m fibrils or r-β2-m monomer (final 0 or 100 μg/ml), or Triton X-100 as a positive control (final 2%) for 15 min (B) or 1 day (C), the fluorescence was measured as described in Materials and Methods. (B, C) β2-m amyloid fibrils did not significantly destruct artificial plasma membranes of LUVs. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05 vs. positive control.</p

Improved artificial intelligence discrimination of minor histological populations by supplementing with color-adjusted images

Abstract Despite the dedicated research of artificial intelligence (AI) for pathological images, the construction of AI applicable to histopathological tissue subtypes, is limited by insufficient dataset collection owing to disease infrequency. Here, we present a solution involving the addition of supplemental tissue array (TA) images that are adjusted to the tonality of the main data using a cycle-consistent generative adversarial network (CycleGAN) to the training data for rare tissue types. F1 scores of rare tissue types that constitute < 1.2% of the training data were significantly increased by improving recall values after adding color-adjusted TA images constituting < 0.65% of total training patches. The detector also enabled the equivalent discrimination of clinical images from two distinct hospitals and the capability was more increased following color-correction of test data before AI identification (F1 score from 45.2 ± 27.1 to 77.1 ± 10.3, p < 0.01). These methods also classified intraoperative frozen sections, while excessive supplementation paradoxically decreased F1 scores. These results identify strategies for building an AI that preserves the imbalance between training data with large differences in actual disease frequencies, which is important for constructing AI for practical histopathological classification