Possible transmission of leukocyte chemotactic factor 2 amyloidosis after interpopulational liver transplantation

Possible transmission of leukocyte chemotactic factor 2 amyloidosis after interpopulational liver transplantatio

Involvement of Macrophages in the Pathogenesis of Familial Amyloid Polyneuropathy and Efficacy of Human iPS Cell-Derived Macrophages in Its Treatment

<div><p>We hypothesized that tissue-resident macrophages in familial amyloid polyneuropathy (FAP) patients will exhibit qualitative or quantitative abnormalities, that may accelerate transthyretin (TTR)-derived amyloid deposition. To evaluate this, we examined the number and subset of tissue-resident macrophages in heart tissue from amyloid-deposited FAP and control patients. In both FAP and control patients, tissue-resident macrophages in heart tissue were all Iba⁺/CD163⁺/CD206⁺ macrophages. However, the number of macrophages was significantly decreased in FAP patients compared with control patients. Furthermore, the proportion of intracellular TTR in CD14⁺ monocytes was reduced in peripheral blood compared with healthy donors. Based on these results, we next examined degradation and endocytosis of TTR in human induced pluripotent stem (iPS) cell-derived myeloid lineage cells (MLs), which function like macrophages. iPS-MLs express CD163 and CD206, and belong to the inhibitory macrophage category. In addition, iPS-MLs degrade both native and aggregated TTR in a cell-dependent manner <i>in vitro</i>. Further, iPS-MLs endocytose aggregated, and especially polymerized, TTR. These results suggest that decreased tissue-localized macrophages disrupt clearance of TTR-derived amyloid deposits, leading to progression of a pathological condition in FAP patients. To improve this situation, clinical application of pluripotent stem cell-derived MLs may be useful as an approach for FAP therapy.</p></div

Clinical Characteristics of FAP ATTR V30M and Control Patients.

<p>Clinical Characteristics of FAP ATTR V30M and Control Patients.</p

Intracellular TTR immunoreactivity in iPS-MLs.

<p>iPS-MLs or control SH-SY5Y cells (1 × 10⁴, 5 × 10⁴, or 1× 10⁵ cells/well) were cultured with native wild-type, wild-type-derived aggregated, native mutated, and mutated-derived aggregated TTR as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163944#pone.0163944.g005" target="_blank">Fig 5</a>. Cultured iPS-MLs and SH-SY5Y cells were stained with TTR antibody by immunohistochemistry. (A) All slides shown are representative for each group. Arrows indicate intracellular TTR. Scale bars: 50 μm. (B-C) Five visual fields in each stained section were randomly chosen, and the number of TTR⁺ cells counted by two observers. Bar graphs show the frequency of TTR⁺ cells in (B) iPS-MLs and (C) SH-SY5Y cells. The proportion of repeated count TTR⁺ cells were analyzed using the mixed model, with *<i>p</i> < 0.05 and **<i>p</i> < 0.01 indicating a significant difference.</p

iPS-MLs degrade aggregated TTR.

<p>(A-H) iPS-MLs or control SH-SY5Y cells (1 × 10⁴, 5 × 10⁴, or 1× 10⁵ cells/well) were cultured with native wild-type, mutated TTR, wild-type-derived, and mutated-derived aggregated TTR for 3 days. Detection of TTR in each culture supernatant was determined by ELISA (A-D) or western blot analysis (E-H). (A-B and E-F) Native wild-type and wild-type-derived aggregated TTR. (C-D and G-H) Native mutated and mutated-derived aggregated TTR. (A-D) Tukey post-hoc test after separate two-way repeated-measures analysis of variance. **<i>p</i> < 0.01. Data are representative of two independent experiments. (E-H) Black arrows and white arrowheads indicate full-length TTR and truncated TTR, respectively. Square brackets indicate polymerized TTR. Mutated TTR represents V30M TTR.</p

Decreased frequency of intracellular TTR in CD14⁺ monocytes from FAP ATTR V30M patients.

<p>CD14^<b>+</b> monocytes were isolated from PBMC of HD (<i>n</i> = 7) or FAP ATTR V30M patients (<i>n</i> = 6) using Ficoll-Paque and magnet bead-conjugated anti-human CD14 antibodies. (A, B) Separated CD14^<b>+</b> monocytes were stained with an anti-human TTR antibody. Five visual fields in each stained section were randomly chosen, and the number of TTR-positive CD14^<b>+</b> monocytes counted by three independent observers. The average count number per visual field is shown. The bar graph (A) shows the frequency of TTR⁺ cells in CD14⁺ monocytes. Photographs (B) show representative data for each group. Scale bars indicate 25 μm. (A, B) The proportion of repeated count TTR⁺ CD14⁺ monocytes were analyzed using the mixed model, with *<i>p</i> < 0.05 indicating a significant difference.</p

Morphology and expression of inhibitory macrophage markers in iPS-MLs.

<p>(A) iPS-ML morphology is shown. (B) Flow cytometry analysis shows expression of myeloid markers including CD45, CD33, CD14, and CD11b on iPS-MLs. Inhibitory macrophage markers, CD163 and CD206, were identified on iPS-MLs. Staining profiles of specific (black areas) and isotype-matched control (gray lines) monoclonal antibodies are shown. Data are representative of three independent experiments. Scale bars: 100 μm.</p

Histopathological characteristics in FAP ATTR V30M and control patients.

<p>Histopathological findings in heart tissue. HE-stained (A-C and J-L), Congo red-stained (D-F and M-O), and Congo red-stained tissue showing green birefringence under polarizing light (G-I and P-R) in control and FAP ATTR V30M patients. Control patient 1 (A, D, G), patient 2 (B, E, H), and patient 3 (C, F, I); and FAP patient 1 (J, M, P), patient 2 (K, N, Q), and patient 3 (L, O, R). Scale bars: 200 μm. Representative cases are shown.</p

Lower number of tissue-resident macrophages in FAP ATTR V30M patients.

<p>(A, B) Heart tissue (FAP ATTR V30M patients, <i>n</i> = 16; control patients, <i>n</i> = 11) was stained with macrophage-related (Iba1) and inhibitory macrophage (CD163 and CD206) markers by immunohistochemistry. All slides shown are representative of each group. Lower (A) and higher (B) magnification views are shown. (C-E) Five visual fields in each stained section were randomly chosen, and the number of Iba1, CD163, and CD206-positive cells counted by two independent observers. Graphs show the average count number per visual field for each marker: Iba1 (C), CD163, (D) and CD206 (E). Repeated count immunostained cells were analyzed using the generalized Poisson mixed model, with *<i>p <</i> 0.05 and **<i>p</i> < 0.01 indicating significant differences. (F) Double immunohistochemical staining of Iba1 and CD206 in heart tissue of FAP ATTR V30M patients. Black arrows show double-immunostained cells. Bars indicate 200 μm (A) and 50 μm in (B, F).</p

Clinicopathological and biochemical findings of thyroid amyloid in hereditary transthyretin amyloidosis with and without liver transplantation

<p>Hereditary transthyretin (TTR) amyloidosis is a fatal disease causing systemic organ dysfunctions. Histopathological studies revealed that thyroid glands are major target tissues. However, details about thyroid functions remain to be fully elucidated in this disease. For patient treatment, liver transplantation (LT) reportedly prolongs patient survival, but thyroid gland function after LT still remains poorly understood. In this study, we investigated the thyroid functions in 101 patients with hereditary TTR amyloidosis and the effects of LT on thyroid functions in those patients. In addition, we investigated histopathological and biochemical findings of thyroid specimens obtained at autopsy. Disease duration and age at examination inversely correlated with serum levels of free triiodothyronine (fT3) in hereditary TTR amyloidosis. On the contrary, in patients who underwent transplantation, time from disease onset to transplantation and age at transplantation clearly correlated with serum fT3and thyroid stimulating hormone (TSH) levels. In autopsy studies, amounts of thyroid amyloid deposits in patients with transplantation were significantly lower than those in patients without transplantation. Mass spectrometric analyzes also revealed that proportions of wild-type (WT) TTR in thyroid amyloid deposits in patients with hereditary TTR amyloidosis who underwent transplantations were higher than those in patients without transplantation. Thyroid hormone functions may diminish according to the disease progression. LT could prevent thyroid dysfunction in hereditary TTR amyloidosis.</p