Transthyretin (TTR) is a homotetrameric protein mainly synthesised by the liver and the choroid plexus whose function is to carry the thyroid hormone thyroxine and the retinol-binding protein bound to retinol in plasma and cerebrospinal fluid. When the stability of the tetrameric structure is lost, it breaks down, paving the way for the aggregation of TTR monomers into insoluble fibrils leading to transthyretin (ATTR) amyloidosis, a progressive disorder mainly affecting the heart and nervous system. Several TTR gene mutations have been characterised as destabilisers of TTR structure and are associated with hereditary forms of ATTR amyloidosis. The reason why also the wild-type TTR is intrinsically amyloidogenic in some subjects is largely unknown. The aim of the review is to give an overview of the TTR biological life cycle which is largely unknown. For this purpose, the current knowledge on TTR physiological metabolism, from its synthesis to its catabolism, is described. Furthermore, a large section of the review is dedicated to examining in depth the role of mutations and physiological ligands on the stability of TTR tetramers.

Cell-derived extracellular vesicles (EVs) found in the circulation and body fluids contain biomolecules that could be used as biomarkers for lung and other diseases. EVs from bronchoalveolar lavage (BAL) might be more informative of lung abnormalities than EVs from blood, where information might be diluted. To compare EVs’ characteristics in BAL and blood in smokers with and without COPD. Same-day BAL and blood samples were obtained in 9 nonsmokers (NS), 11 smokers w/o COPD (S), and 9 with COPD (SCOPD) (FEV1: 59 ± 3% pred). After differential centrifugation, EVs (200–500 nm diameter) were identified by flow cytometry and labeled with cell-type specific antigens: CD14 for macrophage-derived EVs, CD326 for epithelial-derived EVs, CD146 for endothelial-derived EVs, and CD62E for activated-endothelial-derived EVs. In BAL, CD14-EVs were increased in S compared to NS [384 (56–567) vs. 172 (115–282) events/μL; p = 0.007] and further increased in SCOPD [619 (224–888)] compared to both S (p = 0.04) and NS (p < 0.001). CD326-EVs were increased in S [760 (48–2856) events/μL, p < 0.001] and in SCOPD [1055 (194–11,491), p < 0.001] when compared to NS [15 (0–68)]. CD146-EVs and CD62E-EVs were similar in the three groups. In BAL, significant differences in macrophage and epithelial-derived EVs can be clearly detected between NS, S and SCOPD, while these differences were not found in plasma. This suggests that BAL is a better medium than blood to study EVs in lung diseases.

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Background and aims Wild type transthyretin (TTR) cardiac amyloidosis (ATTRwt-CA) is caused by the misfolding, aggregation and tissue deposition of native TTR, leading to cardiac structural remodeling. In the last decade, the validation of non-invasive diagnostic approaches and the development of novel therapeutic options targeting the amyloidogenic cascade, such as the TTR stabilizer tafamidis, have dramatically changed the epidemiology and the prognosis of the disease. Nonetheless, the basic pathophysiological mechanisms underlying TTR misfolding and aggregation are still poorly understood. We aimed to characterize circulating TTR forms in plasma samples of ATTRwt-CA patients before and after treatment with tafamidis through a native electrophoretic method. Methods Plasma samples from 6 male patients with ATTRwt amyloidosis (median age 82 years, IQR 80-83), collected before (T0) and during tafamidis treatment, and from 6 healthy controls were collected. Plasma samples were separated on a native 4–20% Tris-Gly polyacrylamide gel. Western blot analysis was performed with anti-TTR or anti-retinol-binding protein (RBP) antibodies. Proteins were detected by Clarity ECL substrate. Results Circulating TTR forms were qualitatively similar between ATTRwt-CA patients at T0 and controls. In both groups, the most represented forms were TTR dimers or trimers (∼37-50 kDa), TTR tetramers complexed with RBP in 1:1 ratio (∼80 kDa) or 1:2 ratio (∼100 kDa), and high molecular weight (MW) aggregates (&gt;150 kDa). Neither TTR monomers nor tetramers could be detected. RBP protein was detectable in association with TTR tetramers and some of the higher MW fractions (∼150 kDa, &gt;250 kDa). Following tafamidis treatment, all ATTRwt-CA patients displayed a progressive increase of the intensity of the band corresponding to TTR-RBP complexes, in agreement with the a stabilizing action on TTR tetramers. Interestingly, dimers and trimers, detectable at T0, were progressively lost during tafamidis treatment. Conclusions TTR tetramer exists only complexed with RBP and in equilibrium with low and high MW forms, without apparent qualitative differences between ATTR-CA and healthy controls. Tafamidis increases circulating TTR tetramers complexed with RBP. Evaluation of TTR isoform may prove useful as a circulating biomarker for the assessment of response to treatment in patients with ATTRwt-CA.

Introduction Transthyretin (TTR) is a homotetrameric 55-KDa plasma protein that transports thyroxin and retinol complexed to retinol-binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid (ATTR) representing one of the most frequent forms of amyloidosis in elderly. Aim of this study is to develop a native electrophoretic method to characterize circulating TTR in plasma samples of ATTR patients before and during treatment with tafamidis, a TTR stabilizer. Methods Plasma from ATTR patients (n=6), collected before (T0) and during tafamidis treatment, and plasma of healthy controls (n=6) were obtained from Fondazione Toscana G. Monasterio (Pisa, Italy). Plasma samples were separated on a native 4–20% Tris-Gly polyacrylamide gel. Western blot analysis was performed with anti-TTR (DAKO) or anti-RBP (Siemens Healthineer) antibodies. Proteins were detected by Clarity ECL substrate (BioRad). Results Circulating forms of TTR were qualitatively similar between ATTRwt patients at T0 and controls. In both groups, the most represented forms were: TTR dimers or trimers (∼37-50 kDa), TTR tetramers complexed with RBP protein in 1:1 ratio (∼80 kDa,) or 1:2 ratio (∼100 kDa), and high molecular weight (MW) aggregates (&gt;150 kDa). Neither TTR monomers nor the tetramers were visible. RBP protein was detectable in association with TTR tetramers and some of the higher MW fractions (∼150 kDa, &gt;250 kDa). Following tafamidis treatment, all ATTRwt patients displayed a progressive increase of the intensity of the band corresponding to TTR-RBP complexes, in agreement with the drug stabilizing action on TTR tetramers. Interestingly dimers and trimers, detectable at T0, were progressively lost during tafamidis treatment. Conclusions The native electrophoretic allowed us to detect several circulating TTR fractions. Data suggest a similar pattern of circulating TTR both in patients and healthy control: TTR tetramer exists only complexed with RBP and in equilibrium with low and high MW forms. Furthermore, the method allowed to appreciate the stabilizing effect of tafamidis on circulating TTR tetramers complexed with RBP. The study of circulating TTR fractions can expand our knowledge on mechanisms triggering its destabilization even when not mutated.