Early skin denervation in hereditary and iatrogenic transthyretin amyloid neuropathy.

OBJECTIVE: To elucidate early skin denervation in hereditary transthyretin (TTR) amyloidosis and iatrogenic TTR amyloidosis. METHODS: We investigated intraepidermal nerve fiber density (IENFD) and clinical findings in 32 patients with hereditary TTR amyloidosis, 11 asymptomatic mutation carriers, 6 patients with iatrogenic TTR amyloidosis, and 23 healthy volunteers. RESULTS: IENFD values were reduced in patients with the V30M mutation (1.9 ± 2.1 per 1 mm), patients with non-V30M mutations (5.8 ± 3.2 per 1 mm), and patients with iatrogenic TTR amyloidosis (3.5 ± 1.8 per 1 mm) compared with healthy volunteers (11.8 ± 3.2 per 1 mm) (p < 0.01). Skin denervation also occurred, even in presymptomatic V30M mutation carriers (5.0 ± 2.2 per 1 mm). The IENFD was correlated with disease duration (ρ = -0.533, p = 0.002) and various peripheral neuropathy parameters such as sensory impairment in the Kumamoto clinical score (ρ = -0.575, p = 0.001), heat-pain detection threshold (ρ = -0.704, p < 0.001), and sural sensory nerve action potential (ρ = 0.481, p = 0.005). TTR amyloid deposits frequently occurred in connective tissues and vessels of the dermal reticular layer in patients with hereditary TTR amyloidosis and those with iatrogenic TTR amyloidosis. CONCLUSIONS: Patients with hereditary TTR amyloidosis and those with iatrogenic TTR amyloidosis may show early skin denervation even in the presymptomatic stage. IENFD may thus be useful for early diagnosis and may serve as a biomarker in clinical trials for hereditary and iatrogenic TTR amyloidosis.

Inflammatory state exists in familial amyloid polyneuropathy that may be triggered by mutated transthyretin.

The relationship between familial amyloid polyneuropathy (FAP), which is caused by mutated transthyretin (TTR), and inflammation has only recently been noted. To determine whether inflammation is present in FAP carriers and patients, serum interleukin (IL)-6 concentration in 57 healthy donors (HD), 21 FAP carriers, and 66 FAP patients was examined, with the relationship between IL-6 and TTR assessed in each group by multiple regression analysis and structural equation models (SEM). Compared with HD, IL-6 concentration was elevated in FAP carriers (p = 0.001, 95% CI 0.398-1.571) and patients (p = 0.002, 95% CI 0.362-1.521). Further, SEM indicated a positive relationship between IL-6 and TTR in FAP carriers (p = 0.010, 95% CI 0.019-0.140), but not in HD and FAP patients. In addition, we determined whether TTR induces production of pro-inflammatory cytokines ex vivo. HD-derived CD14 + monocytes and induced pluripotent stem cell-derived myeloid lineage cells from a HD and FAP patient dose-dependently produced IL-6 under mutated and aggregated TTR conditions, compared with wild-type TTR. In conclusion, FAP carriers and patients are in an inflammatory state, with the presence of mutated TTR being a trigger of inflammation, especially in FAP carriers.

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

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.

Novel Antibody for the Treatment of Transthyretin Amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is a systemic amyloidosis mainly caused by amyloidogenic transthyretin (ATTR). This incurable disease causes death ∼10 years after onset. Although it has been widely accepted that conformational change of the monomeric form of transthyretin (TTR) is very important for amyloid formation and deposition in the organs, no effective therapy targeting this step is available. In this study, we generated a mouse monoclonal antibody, T24, that recognized the cryptic epitope of conformationally changed TTR. T24 inhibited TTR accumulation in FAP model rats, which expressed human ATTR V30M in various tissues and exhibited non-fibrillar deposits of ATTR in the gastrointestinal tracts. Additionally, humanized T24 (RT24) inhibited TTR fibrillation and promoted macrophage phagocytosis of aggregated TTR. This antibody did not recognize normal serum TTR functioning properly in the blood. These results demonstrate that RT24 would be an effective novel therapeutic antibody for FAP.

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

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 in vitro. 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.

Rapid detection of wild-type and mutated transthyretins.

BACKGROUND: Familial amyloid polyneuropathy is caused by a variant transthyretin, which is a serum protein secreted by the liver. We previously reported that mutated transthyretins were detected in serum samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The aim of this study was to evaluate the clinical usefulness of SELDI-TOF MS for diagnosis of transthyretin-related amyloidosis. METHODS: We used 106 serum samples obtained from patients who were clinically suspected of having amyloidosis between February 2011 and April 2014. SELDI-TOF MS allowed analysis for transthyretin via a 3-h one-step procedure. RESULTS: Of the 106 patients, 51 are transthyretin amyloidosis. Mutated transthyretins were detected in serum samples from 30 of 51 patients with transthyretin amyloidosis. The results of genetic analysis showed that all of those patients had mutations in the transthyretin gene. For all 18 patients with senile systemic amyloidosis of 51 patients with transthyretin amyloidosis, SELDI-TOF MS detected only wild-type transthyretin peaks, not mutated transthyretin peaks. CONCLUSION: SELDI-TOF MS is a clinically useful tool for diagnosis of transthyretin-related amyloidosis.

Knee osteoarthritis associated with different kinds of amyloid deposits and the impact of aging on type of amyloid.

Amyloidosis is a protein conformational disorder in which amyloid fibrils accumulate in the extracellular space and induce organ dysfunction. Recently, two different amyloidogenic proteins, transthyretin (TTR) and apolipoprotein A-I (Apo A-I), were identified in amyloid deposits in knee joints in patients with knee osteoarthritis (OA). However, clinicopathological differences related to those two kinds of amyloid deposits in the knee joint remain to be clarified. Here, we investigated the clinicopathological features related to these knee amyloid deposits associated with knee OA and the biochemical characteristics of the amyloid deposits. We found that all of our patients with knee OA had amyloid deposits in the knee joints, especially in the meniscus, and those deposits were primarily derived from TTR and/or Apo A-I. Some patients with knee OA, however, had unclassified amyloid deposits. One of our interesting observations concerned the different effects of aging on each type of amyloid formed. The frequency of formation of ATTR deposits clearly increased with age, but that of AApo A-I deposits decreased. Furthermore, we found that ∼16% of patients with knee OA developed ATTR/AApo A-I double deposits in the meniscus. Amyloid deposition may therefore be a common histopathological feature associated with knee OA. Also, aging may induce ATTR formation in the knee joint in elderly patients with knee OA, whereas AApo A-I formation may be inversely correlated with age.

C-terminal unfolding of an amyloidogenic ß2-microglobulin fragment: ΔN6ß2-microglobulin.

OBJECTIVES: A ß2-microglobulin (ß2m) fragment that lacks the first six amino acids, i.e., ΔN6ß2-microglobulin (ΔN6ß2m), is an endogenous, proteolytically derived, amyloidogenic fragment of ß2m, the precursor protein in Aß2M amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of ß2m, in this study we aimed to investigate conformational characteristics of ΔN6ß2m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of ß2m with C-terminal unfolding (ß2m92-99) in serum samples from 10 patients undergoing hemodialysis (HD). METHODS: We utilized capillary electrophoretic analysis, surface plasmon resonance and enzyme-linked immunosorbent assay. RESULTS AND CONCLUSIONS: We confirmed the normal core structure of ΔN6ß2m with a commercial monoclonal anti-ß2m antibody. In addition, using the specific monoclonal antibody for the C-terminal peptide, i.e. mAb 92-99, we confirmed unfolding in the C-terminal region of ΔN6ß2m. On the basis of these findings, we established an ELISA to measure ß2m92-99 using ΔN6ß2m as a standard molecule in circulation. However, we did not detect ß2m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum.

Novel styrylbenzene derivatives for detecting amyloid deposits.

BACKGROUND: Various styrylbenzene compounds were synthesized and evaluated as mainly Aß amyloid sensors. These compounds, however, cannot be used for detecting amyloid deposition in peripheral nerves because of the inherent sensitivity of the compounds. These compounds often generate false positives especially in the basement membrane of blood vessels in histochemical studies. To overcome these problems, we must first synthesize other styryl compounds for detecting amyloid fibrils in tissues. METHODS: A wide variety of symmetrical and unsymmetrical styrylbenzene derivatives were synthesized and then these compounds were used to detect amyloid fibrils in autopsy and biopsy samples from patients with various systemic and localized forms of amyloidosis such as familial amyloidotic polyneuropathy (FAP), senile systemic amyloidosis (SSA), amyloid A (AA) amyloidosis, localized AL amyloidosis, and Alzheimer's disease. RESULTS: 1-Methoxy-2,5-bis-styrylbenzene and 2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-2,5-bis-styrylbenzene (EEEFSB) detected amyloid fibrils in both in vitro and in vivo histopathological studies. 1-Methoxy-2,5-bis-styrylbenzene also showed a high strength of fluorescence with amyloid deposition in peripheral nerves in a patient with FAP. CONCLUSIONS: 1-Methoxy-2,5-bis-styrylbenzene and EEEFSB may prove a useful tool for diagnosing amyloidosis, not only in a histochemical study but also in whole body amyloid positron emission tomography (PET) imaging.

Effect of age and sex differences on wild-type transthyretin amyloid formation in familial amyloidotic polyneuropathy: a proteomic approach.

BACKGROUND: Age and sex differences are closely related to the onset of senile systemic amyloidosis (SSA) caused by wild-type (WT) transthyretin (TTR). However, the effects of these differences on the amyloid formation mechanism in familial amyloid polyneuropathy (FAP) caused by variant TTR, have remained unclear. To elucidate age and sex differences in FAP, we investigated biochemical characteristics of amyloid deposits in different tissue sites of FAP by proteomic analysis. METHODS: We used shotgun liquid chromatography/tandem mass spectrometry to analyze the proportions of variant and WT TTR in amyloid deposits in different tissues, such as cardiac, kidney, peripheral nerves, and gastrointestinal tissues, from 23 autopsied FAP cases. RESULTS AND CONCLUSIONS: The analysis revealed a highly significant correlation between the proportion of WT TTR and age at autopsy in cardiac tissues, whereas the analysis indicated no correlation in kidney, peripheral nerves, and gastrointestinal tissues. In addition, we demonstrated age-related significantly increased WT TTR deposits, but not variant TTR deposits, in cardiac tissues of male patients. Taken together, these data suggest that both age and sex differences affect cardiac amyloid formation, mainly derived from WT TTR, in FAP.