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Systemic light chain (LC) amyloidosis (AL) is a disease where organs are damaged by an overload of a misfolded patient-specific antibody-derived LC, secreted by an abnormal B cell clone. The high LC concentration in the blood leads to amyloid deposition at organ sites. Indeed, cryogenic electron microscopy (cryo-EM) has revealed unique amyloid folds for heart-derived fibrils taken from different patients. Here, we present the cryo-EM structure of heart-derived AL amyloid (AL59) from another patient with severe cardiac involvement. The double-layered structure displays a u-shaped core that is closed by a β-arc lid and extended by a straight tail. Noteworthy, the fibril harbours an extended constant domain fragment, thus ruling out the variable domain as sole amyloid building block. Surprisingly, the fibrils were abundantly concatenated with a proteinaceous polymer, here identified as collagen VI (COLVI) by immuno-electron microscopy (IEM) and mass-spectrometry. Cryogenic electron tomography (cryo-ET) showed how COLVI wraps around the amyloid forming a helical superstructure, likely stabilizing and protecting the fibrils from clearance. Thus, here we report structural evidence of interactions between amyloid and collagen, potentially signifying a distinct pathophysiological mechanism of amyloid deposits., (© 2024. The Author(s).)

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 V L domain and stabilise an unexpected partially open LC dimer in which the two V L domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Immunoglobulin light chain amyloidosis (AL) is caused by the aberrant production of amyloidogenic light chains (LC) that accumulate as amyloid deposits in vital organs. Distinct LC sequences in each patient yield distinct amyloid structures. However different tissue microenvironments may also cause identical protein precursors to adopt distinct amyloid structures. To address the impact of the tissue environment on the structural polymorphism of amyloids, we extracted fibrils from the kidney of an AL patient (AL55) whose cardiac amyloid structure was previously determined by our group. Here we show that the 4.0 Å resolution cryo-EM structure of the renal fibril is virtually identical to that reported for the cardiac fibril. These results provide the first structural evidence that LC amyloids independently deposited in different organs of the same AL patient share a common fold., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The Nomenclature Committee of the International Society of Amyloidosis met at the XVIII International Symposium on Amyloidosis in September and virtually in October 2022 with discussions resulting in this upgraded nomenclature recommendation. The nomenclature principles remain unchanged but there is an ongoing discussion regarding the importance and varying nature of intracellular protein aggregates, particularly those associated with neurodegenerative diseases. Six novel proteins were added to the list of human amyloid fibril proteins. Of these, three are polypeptide hormones and two currently utilised peptide drugs, making the number of known iatrogenic amyloid forms four, all appearing as subcutaneous nodules at the injection site. The sixth novel amyloid fibril protein is the transmembrane 106B protein, forming intracellular amyloid fibrils in disorders associated with frontotemporal dementia. The number of known human amyloid fibril proteins is now 42.

Systemic amyloidoses are characterized by the unrelenting deposition of autologous proteins as highly ordered fibrils in target organs. The ensuing, potentially fatal organ dysfunction is the result of the combined damage caused by the proteotoxic effect of prefibrillar species and by the cytotoxicity and the structural alterations produced by the amyloid fibrils. Current therapy is focused on eliminating the amyloid protein, thus extinguishing the amyloid cascade at its origin. While this approach may end the cell damage caused by prefibrillar aggregates and prevent further amyloid accumulation, the noxious effects of the amyloid fibrils persist and may hamper the recovery of organ function, which is the ultimate goal of therapy as it is necessary to improve the quality of life and extend survival. Preclinical studies indicate that the clearance of amyloid deposits can be accelerated by specific antibodies targeting amyloid fibrils that activate complement-mediated macrophages and giant cell phagocytosis, possibly promoting the recovery of organ function. Measuring the therapeutic effect of anti-amyloid agents is still a matter of research. In recent years, several monoclonal antibodies targeting amyloid deposits have been tested in clinical trials with mixed outcomes. Recent encouraging results from phase I/II trials, new anti-amyloid agents, and new antibody engineering offer hope that effective amyloid removal will be accomplished in the near future, accelerating organ recovery and improving quality of life and survival., (© 2022. The Author(s).)

Light-chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full-length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel-based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL λ amyloidosis patient (AL-55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC's dimeric and fibrillar states. All tissues contain fragmented LCs along with the full-length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ-specific, likely due to a complex of proteases. Cleavage sites are concentrated in 'proteolysis-prone' regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo , proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Lavatelli et al.)

The ISA Nomenclature Committee met electronically before and directly after the XVII ISA International Symposium on Amyloidosis, which, unfortunately, had to be virtual in September 2020 due to the ongoing COVID-19 pandemic instead of a planned meeting in Tarragona in March. In addition to confirmation of basic nomenclature, several additional concepts were discussed, which are used in scientific amyloid literature. Among such concepts are cytotoxic oligomers, protofibrils, primary and secondary nucleation, seeding and cross-seeding, amyloid signature proteins, and amyloid plaques. Recommendations for their use are given. Definitions of amyloid and amyloidosis are confirmed. Possible novel human amyloid fibril proteins, appearing as 'classical' in vivo amyloid, were discussed. It was decided to include fibulin-like extracellular matrix protein 1 (amyloid protein: AEFEMP1), which appears as localised amyloid in portal veins. There are several possible amyloid proteins under investigation, and these are included in a new Table.

In light chain amyloidosis (AL), fibrillar deposition of monoclonal immunoglobulin light chains (LCs) in vital organs, such as heart, is associated with their severe dysfunction. In addition to the cellular damage caused by fibril deposition, direct toxicity of soluble prefibrillar amyloidogenic proteins has been reported, in particular, for cardiotoxicity. However, the molecular bases of proteotoxicity by soluble LCs have not been clarified. Here, to address this issue, we rationally engineered the amino acid sequence of the highly cardiotoxic LC H6 by introducing three residue mutations, designed to reduce the dynamics of its native state. The resulting mutant (mH6) is less toxic than its parent H6 to human cardiac fibroblasts and C. elegans. The high sequence and structural similarity, together with the different toxicity, make H6 and its non-toxic designed variant mH6 a test case to shed light on the molecular properties underlying soluble toxicity. Our comparative structural and biochemical study of H6 and mH6 shows closely matching crystal structures, whereas spectroscopic data and limited proteolysis indicate that H6 displays poorly cooperative fold, higher flexibility, and kinetic instability, and a higher dynamic state in its native fold. Taken together, the results of this study show a strong correlation between the overall conformational properties of the native fold and the proteotoxicity of cardiotropic LCs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Transthyretin (TTR) is a tetrameric protein synthesized mostly by the liver. As a result of gene mutations or as an ageing-related phenomenon, TTR molecules may misfold and deposit in the heart and in other organs as amyloid fibrils. Cardiac involvement in TTR-related amyloidosis (ATTR) manifests typically as left ventricular pseudohypertrophy and/or heart failure with preserved ejection fraction. ATTR is an underdiagnosed disorder as well as a crucial determinant of morbidity and mortality, thus justifying the current quest for a safe and effective treatment. Therapies targeting cardiac damage and its direct consequences may yield limited benefit, mostly related to dyspnoea relief through diuretics. For many years, liver or combined heart and liver transplantation have been the only available treatments for patients with mutations causing ATTR, including those with cardiac involvement. The therapeutic options now include several pharmacological agents that inhibit hepatic synthesis of TTR, stabilize the tetramer, or disrupt fibrils. Following the positive results of a phase 3 trial on tafamidis, and preliminary findings on patisiran and inotersen in patients with ATTR-related neuropathy and cardiac involvement, we provide an update on this rapidly evolving field, together with practical recommendations on the management of cardiac involvement., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration., Competing Interests: Conflict of interest statement: G.J.M., N.L.Y., and J.W.K. have submitted a patent application for the small-molecule kinetic stabilizers of Ig light chains.

Systemic light chain amyloidosis (AL)  is a life-threatening disease caused by aggregation and deposition of monoclonal immunoglobulin light chains (LC) in target organs. Severity of heart involvement is the most important factor determining prognosis. Here, we report the 4.0 Å resolution cryo-electron microscopy map and molecular model of amyloid fibrils extracted from the heart of an AL amyloidosis patient with severe amyloid cardiomyopathy. The helical fibrils are composed of a single protofilament, showing typical 4.9 Å stacking and cross-β architecture. Two distinct polypeptide stretches (total of 77 residues) from the LC variable domain (V l ) fit the fibril density. Despite V l high sequence variability, residues stabilizing the fibril core are conserved through different cardiotoxic V l , highlighting structural motifs that may be common to misfolding-prone LCs. Our data shed light on the architecture of LC amyloids, correlate amino acid sequences with fibril assembly, providing the grounds for development of innovative medicines.

Deposition of misfolded proteins as extracellular amyloid aggregates is the pathological hallmark of systemic amyloidoses. Subcutaneous fat acquired by fine needle aspiration is the preferred screening tissue in suspected patients. In this study we employed Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) to investigate human abdominal fat aspirates with the aim of detecting disease-related changes in the molecular structure and composition of the tissue and exploiting the potentiality of the method to discriminate between amyloid-positive and -negative samples. The absorption and second-derivative spectra of Congo Red (CR) positive and CR-negative specimens were analyzed by three multivariate methods in four spectral regions. The proposed ATR-FTIR method is label-free, rapid, and relatively inexpensive and requires minimal sample preparation. We found that the ATR-FTIR approach can differentiate fat aspirates containing amyloid deposits from control specimens with high sensitivity and specificity, both at 100 [89-100]%. It is worth noting that the wavenumbers most important for discrimination indicate that changes both in the protein conformation and in resident lipids are intrinsic features of affected subcutaneous fat in comparison with the CR-negative controls. In this proof of concept study, we show that this approach could be useful for assessing tissue amyloid aggregates and for acquiring novel knowledge of the molecular bases of the disease.

The nomenclature committee of the International Society of Amyloidosis (ISA) meets every second year to discuss and formulate recommendations. The conclusions from the discussion at the XVI International Symposium on Amyloidosis in Kumamoto, Japan, 25-29 March 2018 and afterwards are summarized in this Nomenclature Article. From having recommended the use of the designation "amyloid fibril" for in vivo material only, ISA's nomenclature committee now accepts its use more broadly following the international scientific literature. However, it is important always to stress the origin of the β-fibrils in order to avoid misunderstanding. Given the more broad use of the word "amyloid" several classes of amyloid fibrils may be distinguished. For the medical in vivo situation, and to be included in the amyloid nomenclature list, "amyloid" still means mainly extracellular tissue deposits of protein fibrils, recognized by specific properties, such as green-yellow birefringence after staining with Congo red. It should also be underlined that in vivo amyloid fibrils, in addition to the main protein contain associated compounds, particularly serum amyloid P-component (SAP) and proteoglycans, mainly heparan sulfate proteoglycan. With this definition there are presently 36 human amyloid proteins of which 14 appear only associated with systemic amyloidosis and 19 as localized forms. Three proteins can occur both as localized and systemic amyloidosis. Strictly intracellular aggregates are not included in this list.

The disease causing agent in systemic AL amyloidosis is a monoclonal immunoglobulin free light chain, or fragments thereof, circulating in the blood. It is not surprising, therefore, that measurement of serum free light chains plays a central role in the management of this disorder. In this paper, we review the utility of the serum free light chain assay in the investigation, prognostication and monitoring of AL amyloidosis. Data on the two currently available commercial assays is compared and some practical applications of the assay's use are presented. While there are limitations, it is clear that the availability of the free light chain assay in the laboratory is a major advance and plays an essential role in the management of patients with AL amyloidosis.

Accurate diagnosis of systemic amyloidosis is necessary both for assessing the prognosis and for delineating the appropriate treatment. It is based on histologic evidence of amyloid deposits and characterization of the amyloidogenic protein. We prospectively evaluated the diagnostic performance of immunoelectron microscopy (IEM) of abdominal fat aspirates from 745 consecutive patients with suspected systemic amyloidoses. All cases were extensively investigated with clinical and laboratory data, with a follow-up of at least 18 months. The 423 (56.8%) cases with confirmed systemic forms were used to estimate the diagnostic performance of IEM. Compared with Congo-red-based light microscopy, IEM was equally sensitive (75% to 80%) but significantly more specific (100% vs 80%; P < .001). In amyloid light-chain (AL) amyloidosis, κ cases were more difficult to diagnose (sensitivity 71%), whereas the analysis of abdominal aspirate was informative in only 40% of patients with transthyretin amyloidosis. We found a high prevalence (20%) of a monoclonal component in patients with non-AL amyloidosis, highlighting the risk of misdiagnosis and the need for unequivocal amyloid typing. Notably, IEM identified correctly the specific form of amyloidosis in >99% of the cases. IEM of abdominal fat aspirates is an effective tool in the routine diagnosis of systemic amyloidoses., (© 2015 by The American Society of Hematology.)

Background: Nutrition status was shown to be a prognostic factor in patients with immunoglobulin light-chain amyloidosis (AL). However, malnutrition was associated with cardiac involvement, thus suggesting potential interactions. This study aim was to clarify the association among nutrition status, cardiac stage, and mortality in AL., Methods: One hundred twenty-eight consecutive newly diagnosed, treatment-naïve patients with histologically confirmed AL were enrolled. Anthropometric, biochemical, and clinical variables were assessed., Results: At multivariable Cox proportional hazard analysis, body mass index (BMI) < 22 kg/m(2) (HR = 1.98, 95% CI = 1.09-3.56) and unintentional 6-month weight loss (WL) ≥ 10% (HR = 1.94, 95% CI = 1.00-3.74) resulted in independent predictors of survival after controlling for hematologic response to treatment (HR = 0.27, 95% CI = 0.14-0.53) and cardiac stage (Mayo Clinic stage III, HR = 4.42, 95% CI = 2.61-7.51). There was no effect modification of malnutrition on mortality by cardiac stage (P for interaction = .27). Moderate and severe malnutrition (prevalence: 21.9% and 7.8%, respectively) similarly increased the risk of death (HR = 3.09, 95% CI = 1.75-5.46; 2.88, 95% CI = 1.23-6.72, respectively)., Conclusions: In AL, malnutrition at diagnosis is a frequent comorbidity that affects the prognosis independently of hematologic response to treatment and cardiac stage. Nutrition status should be systematically considered in future intervention trials in AL. Nutrition support trials are warranted., (© 2013 American Society for Parenteral and Enteral Nutrition.)

In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of the MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non-amyloid-affected) counterpart were acquired. Label-free comparison between patients and controls made it possible to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism, and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a priori hypotheses, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases.

This phase II, open-label, single-treatment arm study evaluated the pharmacodynamics, efficacy, and safety of tafamidis in patients with non-Val30Met transthyretin (TTR) amyloidosis. Twenty-one patients with eight different non-Val30Met mutations received 20 mg QD of tafamidis meglumine for 12 months. The primary outcome, TTR stabilization at Week 6, was achieved in 18 (94.7%) of 19 patients with evaluable data. TTR was stabilized in 100% of patients with non-missing data at Months 6 (n = 18) and 12 (n = 17). Exploratory efficacy measures demonstrated some worsening of neurological function. However, health-related quality of life, cardiac biomarker N-terminal pro-hormone brain natriuretic peptide, echocardiographic parameters, and modified body mass index did not demonstrate clinically relevant worsening during the 12 months of treatment. Tafamidis was well tolerated. In conclusion, our findings suggest that tafamidis 20 mg QD effectively stabilized TTR associated with several non-Val30Met variants.

Abstract The clinical phenotype of familial ATTR amyloidosis depends to some extent on the particular mutation, but differences exist also within mutations. We have previously described that two types of amyloid fibril compositions exist among Swedish ATTRV30M amyloidosis patients, one consisting of a mixture of intact and fragmented ATTR (type A) and one consisting of mainly intact ATTR (type B). The fibril types are correlated to phenotypic differences. Patients with ATTR fragments have a late onset and develop cardiomyopathy, while patients without fragments have an early onset and less myocardial involvement. The present study aimed to determine whether this correlation between fibril type and phenotype is valid for familial ATTR amyloidosis in general. Cardiac or adipose tissues from 63 patients carrying 29 different TTR non-V30M mutations as well as 13 Japanese ATTRV30M patients were examined. Fibril type was determined by western blotting and compared to the patients' age of onset and degree of cardiomyopathy. All ATTR non-V30M patients had a fibril composition with ATTR fragments, except two ATTRY114C patients. No clear conclusions could be drawn about a phenotype to fibril type correlation among ATTR non-V30M patients. In contrast, Japanese ATTRV30M patients showed a similar correlation as previously described for Swedish ATTRV30M patients. This study shows that a fibril composition with fragmented ATTR is very common in ATTR amyloidosis, and suggests that fibrils composed of only full-length ATTR is an exception found only in a subset of patients.

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

In AL amyloidosis prognosis depends on the severity of heart dysfunction which is best assessed by natriuretic peptides (BNP and NT-proBNP). However, their clearance relies on glomerular filtration rate (GFR) and their concentration increases with renal failure. We evaluated the diagnostic and prognostic performance of NT-proBNP and BNP in 248 patients with AL amyloidosis with different degrees of renal failure. Patients were grouped according to GFR. Group 1 comprised 109 patients with GFR ≥60 mL/min/1.73 m(2) , Group 2, 77 subjects with GFR <60 and ≥15 mL/min/1.73 m(2) , and Group 3, 62 patients with GFR <15 mL/min/1.73 m(2) . The ability of natriuretic peptides to detect heart involvement and to predict survival in the three groups was assessed. Decreasing eGFR required higher cutoffs of both NT-proBNP and BNP for detecting heart involvement and predicting survival. Both natriuretic peptides were independent prognostic markers in Groups 1 and 2, whereas in Group 3 only BNP independently predicted survival. Natriuretic peptides are powerful and useful markers of cardiac dysfunction and prognosis, provided that eGFR is considered in interpreting their clinical meaning. BNP should be preferred in patients with end-stage renal failure., (Copyright © 2012 Wiley Periodicals, Inc.)

Monoclonal Ig light chains (LC) can be responsible for pathologic conditions in humans, as in systemic amyloid light amyloidosis. Protean clinical manifestations characterize this disorder with the most varied combination of symptoms generated by different degrees of diverse organ involvement. Kidney and heart are most frequently interested, with major heart involvement as the most relevant prognostic factor. The identification of the underlying mechanism involved in organ targeting is of major relevance for the pathobiology of this disorder. To this aim, we characterized the repertoire of variable region germline genes of λ LC preferentially targeting the heart and compared it with the repertoire of LC that do not in a case-control study. We found that the repertoires were highly restricted, showing preferential use of the same few germline genes but with a different frequency pattern. A single gene, IGVL1-44, was found associated with a 5-fold increase in the odds of dominant heart involvement (after adjusting for confounders in a multivariable logistic model). These results support an involvement of LC genetics in the determination of organ targeting. Study of the characteristics of IGVL1-44-LC with, and of the minority without, heart involvement might lead to identification of LC/tissue interactions.

Background: Cardiac biomarkers play a major role in the identification of patients at risk of early death in AL amyloidosis, and a staging system based on amino-terminal pro-natriuretic peptide type-B (NT-proBNP) and troponins (cTn) is used for prognostic stratification. Adrenomedullin is produced by several tissues including the heart, and portends a poor prognosis in heart diseases. We investigated the ability of midregional proadrenomedullin (MR-proADM) to predict early death in AL amyloidosis., Methods: One-hundred and thirty consecutive patients with newly-diagnosed AL amyloidosis were prospectively enrolled. The impact on survival of NT-proBNP, cTnI and MR-proADM was evaluated., Results: The concentration of MR-proADM correlated with systolic and diastolic function, but did not reflect the amount of amyloid deposited in the heart. Moreover, MR-proADM was associated with non-cardiac markers of advanced disease. The staging system based on NT-proBNP and cTnI identified high-risk subjects, but could not discriminate good-risk and intermediate-risk patients. Conversely, a staging system based on MR-proADM and cTnI identified 3 groups with significantly different survivals., Conclusions: Midregional-proADM is a powerful prognostic marker in AL amyloidosis, which may not only reflect cardiac dysfunction but also widespread systemic disease, and can be combined with cTn for detecting patients at risk of early death.

The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate. Two mutations (Δ70-72 and L90P) almost abrogated the lag phase of the aggregation process, three mutations (Δ60-71, L75P, and W50R) significantly accelerated the aggregation rate by 2- to 3-fold, while the remaining three variants (L64P, L60R, and G26R) were not significantly different from the wild type. Therefore, an increase in aggregation propensity cannot explain per se the mechanism of the disease for all the variants. Prediction of the protection factors for hydrogen exchange in the native state of full-length protein reveals, in almost all the variants, an expansion of the conformational fluctuations that could favour the proteolytic cleavage and the release of the amyloidogenic peptide. Such an event seems to be a necessary prerequisite for ApoA-I fibrillogenesis in vivo, but the observed increased aggregation propensity of certain variants can have a strong influence on the severity of the disease, such as an earlier onset and a faster progression., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A system of amyloid fibril nomenclature based on the chemical identity of the amyloid fibril forming protein is recommended. This system has been in use for approximately 40 years, but current literature remains confused with clinical and histochemical designations used when the amyloid disease processes were poorly understood. To be designated an amyloid fibril protein, the protein must occur in tissue deposits and exhibit affinity for Congo red and green birefringence when viewed by polarisation microscopy. Furthermore, the protein must have been unambiguously characterised by protein sequence analysis (DNA sequencing in the case of familial diseases). Current nomenclature lists of 27 human and nine animal fibril proteins are provided together with a list of eight inclusion bodies that exhibit some of the properties of amyloid fibrils.

Deposition of amyloid in the buttock is a rare complication of dialysis related amyloidosis (DRA), but this localization is even rarer in other types of amyloidoses. We report here the clinical, radiological, and biochemical features of a patient who incurred into this complication after 27 years of hemodialysis. Imaging of the amyloid deposition by magnetic resonance imaging (MRI) documents the amyloid infiltration in the muscles of the buttock region and highlights a peculiar feature of amyloid fibrils deposition in the subcutaneous fat. The amyloid deposition is confirmed by biochemical and microscopic analysis of fibrils extracted from a biopsy specimen. Review of literature and the features of this case lead to speculation that the peculiar involvement of the buttock region including muscles and subcutaneous fat in DRA might derive from the propagation of amyloid initially deposited in the hip joint.

Protein misfolding and deposition as amyloid, with consequent tissue damage, plays a key role in the group of diseases generically termed amyloidoses. In the systemic forms, amyloid deposition is widespread and causes severe dysfunction of vital organs. Proteomic analysis, thanks to its versatility and the comprehensiveness of information obtained, is an ideal tool for the study of systemic amyloidoses. It has been successfully employed in the characterization of the circulating amyloidogenic precursors and the analysis of affected tissues, for the diagnostic identification of the fibril components and for characterizing disease-related changes in protein expression. We present the developments in the field of proteomics applied to systemic amyloidoses, and discuss the perspectives opened in the study of these diseases.

In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of alphaB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration.

Objective: To report 8 patients with Sjögren's syndrome (SS) and localized nodular cutaneous amyloidosis and to examine serologic and immunohistologic findings that may link the 2 diseases., Methods: The databases for 3 amyloidosis centers were searched for patients with localized nodular cutaneous amyloidosis and SS. Eight patients with this combination were identified, and clinical, serologic, and histologic parameters were retrospectively evaluated., Results: Among the 8 patients with a clinical diagnosis of SS, 6 fulfilled the American-European Consensus Group criteria for SS. All of the patients were women in whom SS had been diagnosed at a median age of 47 years (range 30-61 years) and amyloid had been diagnosed at a median age of 60 years (range 42-79 years). The presence of the immunoglobulin light chain type of amyloid (AL amyloid) was confirmed in 4 patients. In 3 of these 4 patients as well as 2 other patients, a light chain-restricted plasma cell population was observed near the amyloid deposits. Progression to systemic amyloidosis was not observed in any patient during a median followup of 3.5 years., Conclusion: SS should be considered in patients with cutaneous amyloidosis. The combination of cutaneous amyloidosis and SS appears to be a distinct disease entity reflecting a particular and benign part of the polymorphic spectrum of lymphoproliferative diseases related to SS.

Amyloidosis associated to hemodialysis is caused by persistently high beta(2)-microglobulin (beta(2)m) serum levels. beta(2)m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no beta(2)m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to beta(2)m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of beta(2)m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60-->Gly mutation has a threefold effect: it stabilizes beta(2)m, inhibits beta(2)m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the beta(2)m core; the Trp95-->Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar beta(2)m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in beta(2)m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type beta(2)m towards self-aggregation into amyloid fibrils.

The increasing knowledge of the exact biochemical nature of the localized and systemic amyloid disorders has made a logical and easily understood nomenclature absolutely necessary. Such a nomenclature, biochemically based, has been used for several years but the current literature is still mixed up with many clinical and histochemically based designations from the time when amyloid in general was poorly understood. All amyloid types are today preferably named by their major fibril protein. This makes a simple and rational nomenclature for the increasing number of amyloid disorders known in humans and animals.

The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.

Apolipoprotein A-I, the major structural apolipoprotein of high-density lipoproteins, efficiently protects humans from cholesterol accumulation in tissues; however, it can cause systemic amyloidosis in the presence of peculiar amino acid replacements. The wild-type molecule also has an intrinsic tendency to generate amyloid fibrils that localise within the atherosclerotic plaques. The structure, folding and metabolism of normal apolipoprotein A-I are extremely complex and as yet not completely clarified, but their understanding appears essential for the elucidation of the amyloid transition. We reviewed present knowledge on the structure, function and amyloidogenic propensity of apolipoprotein A-I with the aim of highlighting the possible molecular mechanisms that might contribute to the pathogenesis of this disease. Important clues on apolipoprotein A-I amyloidogenesis may be obtained from classical comparative studies of the properties of the wild-type versus the amyloidogenic counterpart. Additionally, in the case of apoA-I, further insights on the molecular mechanisms underlying its amyloidogenic propensity may derive from comparative studies between amyloidogenic variants and other mutations associated with hypoalphalipoproteinemia without amyloidosis.

Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to beta2-microglobulin derived from fibrils but is not observed in the beta2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar beta2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1-2% of the intact molecule.

We undertook this study to develop uniformly accepted criteria for the definition of organ involvement and response for patients on treatment protocols for immunoglobulin light-chain amyloidosis (AL). A consensus panel was convened comprising 13 specialists actively involved in the treatment of patients with amyloidosis. Institutional criteria were submitted from each, and a consensus was developed defining each organ involved and the criteria for response. Specific criteria have been developed with agreed on definitions of organ and hematologic response as a result of discussions at the 10th International Symposium on Amyloid and Amyloidosis held in Tours, France, April 2004. These criteria now form the working definition of involvement and response for the purposes of future data collection and reporting. We report criteria that centers can now use to define organ involvement and uniform response criteria for reporting outcomes in patients with light-chain AL., (Copyright (c) 2005 Wiley-Liss, Inc.)

Background: The term amyloidosis encompasses a wide group of conditions characterised by the tissue deposition of autologous proteins assembled in homogeneous regularly spaced antiparallel beta strands. The mechanism by which the different proteins gain a conformation, allowing monomers to bind to each other to form the regular amyloid fibril, is under intensive investigation. The discovery that human lysozyme, a protein thoroughly structurally and functionally characterised, can form amyloid fibrils has offered unique opportunities to unveil the molecular mechanisms involved in amyloid formation. Four amyloidogenic mutations have been identified and an apparently non-amyloidogenic polymorphism has been recently described., Results and Conclusions: Lysozyme is well characterised for structure, function, folding dynamics and metabolism and comparative studies are becoming available that highlight pathogenic differences between the wild-type and the amyloidogenic variants. The chemical structure of lysozyme in natural amyloid fibrils was characterised in high detail in the early cases, but it is still lacking in the cases most recently discovered. Amyloidogenic lysozymes represent a prototypic molecule for new pharmaceutical approaches in which the formation of amyloid fibrils is abrogated through a stabilisation of the precursor.

The modern nomenclature of amyloidosis now includes 25 human and 8 animal fibril proteins. To be included in the list, the protein has to be a major fibril protein in extracellular deposits, which have the characteristics of amyloid, including affinity for Congo red with resulting green birefringence. Synthetic fibrils with amyloid properties are best named 'amyloid-like'. With increasing knowledge, however, the borders between different protein aggregates tend to become less sharp.

Atomic force microscopy was employed to study ex vivo amyloid material isolated from the transplanted hearts of two patients affected by systemic amyloidosis caused by the Leu174Ser apolipoprotein A-I variant. The purified material consists of fibrils and globular aggregates. For both patients the same morphological patterns are observed; in addition, fibril diameters obtained for the two patients turn out to be compatible, both in air (2.00+/-0.02 and 2.04+/-0.04 nm) and under liquid (10.7+/-0.4 and 11.3+/-0.5 nm). Fibrils display heterogeneous morphologies, occasionally showing a left-handed twist. Inspection of fibril ends, the study of fibril contour shape and the analysis of partially unfolded fibrils yield independent evidences suggesting that most twisted fibrils are composed of three protofilaments. The size of globular aggregates is the same for both patients (4.4+/-0.4 and 5.1+/-0.5 nm, measured under liquid) and is compatible with the protofilament expected diameter, suggesting that globules may represent protofilament precursors.

Knowledge about the systemic amyloidoses has increased considerably during the last few years. This group of diseases is characterized by great biochemical variability, including at least 11 different amyloid fibril proteins and a remarkable range of clinical manifestations. With the understanding that the pathogenesis is different in the various forms of amyloidosis, it is now being increasingly accepted that an early and accurate diagnosis, including that of the underlying biochemical nature, is crucial for a successful treatment. The elucidation of the molecular mechanisms involved in amyloidogenesis is at the basis of the recent blossoming of new, innovative and more effective therapeutic approaches.

Transthyretin Leu55Pro is one of the most aggressive mutations in familial amyloidotic polyneuropathy, an autosomal dominant disorder characterized by extracellular deposition of fibrillar amyloid protein. This variant has the ability to form fibrils in vitro under physiological conditions (PBS, pH 7.4). We studied by transmission electron microscopy the effect of the drug 4'-iodo-4'-deoxydoxorubicin (I-DOX) on the in vitro assembly of TTR Leu55Pro fibrils by following fibril growth over a 15 day period. Our results showed that I-DOX at a concentration of 10-5 M/100 microg fibrils does not inhibit fibril formation in up to 10 days since fibrils identical to the ones present in the untreated sample were observed. However, after 15 days of treatment, only round particles, resembling soluble native TTR, were observed. We also tested the ability of tetracyclines and nitrophenols to interfere with amyloid fibril formation for 17 days; the group of compounds tested showed fibril disruption activity to different extents: doxycycline and 2,4-dinitrophenol resulted in complete disaggregation of fibrils. The species generated upon I-DOX and tetracyclines treatments were nontoxic, as revealed by the lack of significant caspase-3 activation on a Schwannoma cell line, making them potential therapeutic drugs in TTR-related and other amyloidosis.

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.