The Ultrastructure of Tissue Damage by Amyloid Fibrils.

Amyloidosis is a group of diseases that includes Alzheimer's disease, prion diseases, transthyretin (ATTR) amyloidosis, and immunoglobulin light chain (AL) amyloidosis. The mechanism of organ dysfunction resulting from amyloidosis has been a topic of debate. This review focuses on the ultrastructure of tissue damage resulting from amyloid deposition and therapeutic insights based on the pathophysiology of amyloidosis. Studies of nerve biopsy or cardiac autopsy specimens from patients with ATTR and AL amyloidoses show atrophy of cells near amyloid fibril aggregates. In addition to the stress or toxicity attributable to amyloid fibrils themselves, the toxicity of non-fibrillar states of amyloidogenic proteins, particularly oligomers, may also participate in the mechanisms of tissue damage. The obscuration of the basement and cytoplasmic membranes of cells near amyloid fibrils attributable to an affinity of components constituting these membranes to those of amyloid fibrils may also play an important role in tissue damage. Possible major therapeutic strategies based on pathophysiology of amyloidosis consist of the following: (1) reducing or preventing the production of causative proteins; (2) preventing the causative proteins from participating in the process of amyloid fibril formation; and/or (3) eliminating already-deposited amyloid fibrils. As the development of novel disease-modifying therapies such as short interfering RNA, antisense oligonucleotide, and monoclonal antibodies is remarkable, early diagnosis and appropriate selection of treatment is becoming more and more important for patients with amyloidosis.

Inflammatory profiling of patients with familial amyloid polyneuropathy.

BACKGROUND: Familial amyloid polyneuropathy (FAP) or ATTRv (amyloid TTR variant) amyloidosis is a fatal hereditary disease characterized by the deposition of amyloid fibrils composed of transthyretin (TTR). The current diagnosis of ATTRv relies on genetic identification of TTR mutations and on Congo Red-positive amyloid deposits, which are absent in most ATTRv patients that are asymptomatic or early symptomatic, supporting the need for novel biomarkers to identify patients in earlier disease phases allowing disease control. METHODS: In an effort to search for new markers for ATTRv, our group searched for nine inflammation markers in ATTRv serum from a cohort of 28 Brazilian ATTRv patients. RESULTS: We found that the levels of six markers were increased (TNF-α, IL-1ß, IL-8, IL-33, IFN-ß and IL-10), one had decreased levels (IL-12) and two of them were unchanged (IL-6 and cortisol). Interestingly, asymptomatic patients already presented high levels of IL-33, IL-1ß and IL-10, suggesting that inflammation may take place before fibril deposition. CONCLUSIONS: Our findings shed light on a new, previously unidentified aspect of ATTRv, which might help define new criteria for disease management, as well as provide additional understanding of ATTRv aggressiveness.

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.

Differential expression of Cathepsin E in transthyretin amyloidosis: from neuropathology to the immune system.

BACKGROUND: Increasing evidence supports a key role for inflammation in the neurodegenerative process of familial amyloidotic polyneuropathy (FAP). While there seems to be an overactivation of the neuronal interleukin-1 signaling pathway, the immune response is apparently compromised in FAP. Accordingly, little immune cell infiltration is observed around pre-fibrillar or fibrillar amyloid deposits, with the underlying mechanism for this phenomenon remaining poorly understood. Cathepsin E (CtsE) is an important intermediate for antigen presentation and chemotaxis, but its role in the pathogenesis of FAP disease remains unknown. METHODS: In this study, we used both mouse primary macrophages and in vivo studies based on transgenic models of FAP and human samples to characterize CtsE expression in different physiological systems. RESULTS: We show that CtsE is critically decreased in bone marrow-derived macrophages from a FAP mouse model, possibly contributing for cell function impairment. Compromised levels of CtsE were also found in injured nerves of transgenic mice and, most importantly, in naïve peripheral nerves, sensory ganglia, murine stomach, and sural nerve biopsies derived from FAP patients. Expression of CtsE in tissues was associated with transthyretin (TTR) deposition and differentially regulated accordingly with the physiological system under study. Preventing deposition with a TTR small interfering RNA rescued CtsE in the peripheral nervous system (PNS). In contrast, the expression of CtsE increased in splenic cells (mainly monocytes) or peritoneal macrophages, indicating a differential macrophage phenotype. CONCLUSION: Altogether, our data highlights the potential of CtsE as a novel FAP biomarker and a possible modulator for innate immune cell chemotaxis to the disease most affected tissues-the peripheral nerve and the gastrointestinal tract.

Familial amyloidotic polineuropathy and systemic lupus.

Familial amyloidotic polineuropathy is a genetic disorder, leading to systemic amyloid deposits, manifested as sensory-motor and autonomic neuropathy. In the Portuguese classical form, the disease is evident at a young age, and causes death if no specific treatment is received. Variability in penetrance, age of onset and clinical course has been published; environmental and genetic factors are believed to contribute to this variability. The authors report a case of a 51-year-old white female, with a medical history of acquired angioedema, late-onset familial amyloidotic polineuropathy and systemic lupus erythemathosus. The authors consider that these associated diseases could modulate their expression.

Pathological review of cardiac amyloidosis using autopsy cases in a single Japanese institution.

AIM: Amyloidosis is a systemic or localized disease of protein deposition characterized by amorphous eosinophilic morphology and positivity of Congo Red staining. The typing of amyloidosis is becoming increasingly important because therapeutic agents for each amyloidosis type have been developed. Herein, the authors review the autopsy cases at an institution to reveal the putative Japanese characteristics of each amyloidosis type and evaluate the clinicopathological significance of each type. MATERIALS AND METHODS: A total of 131 autopsy cases of systemic and localized amyloidosis were retrieved for classification by immunohistochemistry. Immunohistochemistry for transthyretin, amyloid A (AA), immunoglobulin light-chain kappa and lambda, and ß2-microglobulin was performed for all cases. RESULTS: The 131 amyloidosis cases were classified as follows: 71 cases (54.2%) of transthyretin amyloidosis, 32 cases (24.4%) of AA amyloidosis, 8 cases (6.1%) of light-chain amyloidosis, and 5 cases (3.8%) of ß2-microglobulin amyloidosis, along with 15 equivocal cases (11.5%). All cases showed myocardial involvement of amyloidosis. Histopathologically, the transthyretin type was significantly associated with the interstitial and nodular patterns, and with the absence of the perivascular and endocardial patterns. The AA type was significantly associated with the perivascular and endocardial patterns, and with the absence of the nodular pattern. CONCLUSION: The authors revealed the putative characteristics of cardiac amyloidosis in Japan by using autopsy cases. About 90% of amyloidosis cases were successfully classified using only commercially available antibodies.

The Expression of Chemokines Is Downregulated in a Pre-Clinical Model of TTR V30M Amyloidosis.

Inflammation is a hallmark of several neurodegenerative disorders including hereditary amyloidogenic transthyretin amyloidosis (ATTRv). ATTRv is an autosomal dominant neurodegenerative disorder with extracellular deposition of mutant transthyretin (TTR) aggregates and fibrils, particularly in nerves and ganglia of the peripheral nervous system. Nerve biopsies from ATTRv patients show increased cytokine production, but interestingly no immune inflammatory cellular infiltrate is observed around TTR aggregates. Here we show that as compared to Wild Type (WT) animals, the expression of several chemokines is highly downregulated in the peripheral nervous system of a mouse model of the disease. Interestingly, we found that stimulation of mouse Schwann cells (SCs) with WT TTR results in the secretion of several chemokines, a process that is mediated by toll-like receptor 4 (TLR4). In contrast, the secretion of all tested chemokines is compromised upon stimulation of SCs with mutant TTR (V30M), suggesting that V30M TTR fails to activate TLR4 signaling. Altogether, our data shed light into a previously unappreciated mechanism linking TTR activation of SCs and possibly underlying the lack of inflammatory response observed in the peripheral nervous system of ATTRv patients.

Underlying Immune Disorder May Predispose Some Transthyretin Amyloidosis Subjects to Inotersen-Mediated Thrombocytopenia.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 109/L in 50% and ≥100 × 109/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 109/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR (n = 17 placebo; n = 31 inotersen) and OLE (n = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.

Prevalence of Monoclonal Gammopathy in Wild-Type Transthyretin Amyloidosis.

OBJECTIVE: To evaluate the prevalence of monoclonal gammopathy (MG) in patients with wild-type transthyretin amyloidosis (ATTRwt) (formerly known as senile amyloidosis). PATIENTS AND METHODS: We retrospectively analyzed the serum protein electrophoresis and serum immunofixation results, free light chain (FLC) levels, and renal function of 113 consecutive patients with ATTRwt seen at the Brigham and Women's Hospital's Cardiac Amyloidosis Program between February 21, 2006, and November 9, 2016. Monoclonal gammopathy was defined as a monoclonal protein present in the serum. Light chain MG was defined as an abnormal serum FLC κ/λ ratio with an elevated FLC level in the absence of a monoclonal protein. In patients with renal dysfunction, the renal FLC reference range was used. RESULTS: The mean age of the population was 75 years, 3 of the 113 patients (3%) were female, and 110 (97%) were white. Monoclonal gammopathy was present in 26 patients (23%), 24 of whom had monoclonal protein present and 2 others who met criteria for light chain MG. Most clones (12 of 20 [60%]) were λ restricted. Another 7 patients had an abnormal FLC κ/λ ratio in the setting of renal dysfunction. CONCLUSION: In this study, MG was present in 23% of patients with ATTRwt. The finding of MG or an abnormal FLC κ/λ ratio in an elderly man may cause diagnostic confusion during subtyping of amyloidosis. A high degree of clinical suspicion for ATTRwt and precise tissue typing using mass spectrometry may overcome such diagnostic challenges.

Light-chain cardiac amyloidosis.

Cardiac amyloidosis is an underrecognized condition, in which delays to diagnosis have great implications on management options, prognosis, and morbidity. Once cardiac tissue is infiltrated by amyloid fibrils, there is a cascade of pathologic changes that can display an array of clinical manifestations, from impaired relaxation of the ventricular myocardium to severe restrictive disease or even progressive systolic heart failure. Management is guided not only by recognizing the subtype of amyloidosis (primary, hereditary, and wild-type transthyretin amyloidosis), but also the clinical stage of the disease. It is important for those managing such patients to understand and differentiate disease associated with fibrils composed of transthyretin vs light-chain proteins. Kappa- and lambda-light chains of primary amyloidosis are particularly toxic to myocytes, leading to accelerated clinical illness in the face of intolerance to treatment and poor survival. Limitations to treatment of primary cardiac amyloidosis are related to multiorgan dysfunction and the inability to tolerate appropriate chemotherapy. Bortezomib, a selective protease inhibitor, has been shown to be and an effective and tolerable option for those with myocardial amyloid infiltration. Standard goal-directed optimal medical management for cardiomyopathy (such as beta-blockers and ace inhibitors) does not offer a survival benefit with cardiac amyloidosis, and often is associated with adverse effects. Despite advances in treatment of advanced heart failure therapy, end-stage cardiomyopathy in the setting of amyloidosis is not well stabilized by inotropes or mechanical circulatory support, and offers restricted candidacy for heart transplantation. We review the salient features of cardiac amyloidosis to help general practitioners and subspecialists manage this unique clinical condition.

Case Report of an ABO-Incompatible Living-Donor Liver Transplant for a Familial Amyloid Polyneuropathy Patient.

Liver transplant is a treatment for familial amyloid polyneuropathy. Few cases of ABO-incompatible living-donor liver transplant for familial amyloid polyneuropathy exist. The outcome of an ABO-incompatible living-donor liver transplant has improved recently, using local infusion therapy and rituximab prophylaxis. Here, we describe a successful ABO-incompatible living-donor liver transplant in a patient with familial amyloid polyneuropathy in whom disease progression ceased at 2 years' follow-up. Additionally, no evidence of acute or chronic rejection, or adverse events of the immunosuppressive therapy, was seen. As a postoperative complication, fatty changes in the grafted liver because of malnutrition or adverse events of corticosteroids were confirmed by a liver biopsy taken early after transplant. The main cause of malnutrition was considered to be gastrointestinal dysfunction caused by familial amyloid polyneuropathy. Therefore, before deterioration of digestive function, liver transplants should be considered for familial amyloid polyneuropathy. This case suggests that an ABO-incompatible living-donor liver transplant may provide greater opportunities for familial amyloid polyneuropathy patients.

Optimization of Serum Immunoglobulin Free Light Chain Analysis for Subclassification of Cardiac Amyloidosis.

Accurate and rapid classification of cardiac amyloidosis is important for patient management. We have optimized the use of serum free light chain kappa and lambda values to differentiate immunoglobulin light chain amyloid (AL) amyloidosis from transthyretin amyloid and amyloid A using 85 cases of tissue-proven cardiac amyloidosis, in which there was direct classification of amyloidosis by mass spectrometry or immunofluorescence. The serum free light chain kappa/lambda ratios were non-overlapping for the three major groups: AL-lambda (0.01-0.41, n = 30), non-AL (0.52-2.7, n = 43), and AL-kappa (6.7-967, n = 12). A kappa/lambda ratio value between 0.5 and 5.0 had 100 % sensitivity and 100 % specificity for distinguishing AL amyloidosis from non-AL amyloidosis. This optimized range for serum light chain kappa/lambda ratio provides extremely robust classification of cardiac amyloidosis. Cases of cardiac amyloidosis in which the serum kappa/lambda free light chain ratio falls close to these new cutoff values may benefit most from direct amyloid subtyping.

Clinical features and outcomes of systemic amyloidosis with gastrointestinal involvement: a single-center experience.

BACKGROUND/AIMS: The gastrointestinal (GI) tract often becomes involved in patients with systemic amyloidosis. As few GI amyloidosis data have been reported, we describe the clinical features and outcomes of patients with pathologically proven GI amyloidosis. METHODS: We identified 155 patients diagnosed with systemic amyloidosis between April 1995 and April 2013. Twenty-four patients (15.5%) were diagnosed with GI amyloidosis using associated symptoms, and the diagnoses were confirmed by direct biopsy. RESULTS: Among the 24 patients, 20 (83.3%) had amyloidosis light chain (AL), three (12.5%) had amyloid A, and one (4.2%) had transthyretin-related type amyloidosis. Their median age was 57 years (range, 37 to 72), and 10 patients were female (41.7%). The most common symptoms of GI amyloidosis were diarrhea (11 patients, 45.8%), followed by anorexia (nine patients, 37.5%), weight loss, and nausea and/or vomiting (seven patients, 29.2%). The histologically confirmed GI tract site in AL amyloidosis was the stomach in 11 patients (55.0%), the colon in nine (45.0%), the rectum in seven (35.0%), and the small bowel in one (5.0%). Patients with GI involvement had a greater frequency of organ involvement (p = 0.014). Median overall survival (OS) in patients with GI involvement was shorter (7.95 months; range, 0.3 to 40.54) than in those without GI involvement (15.84 months; range, 0.0 to 114.53; p = 0.069) in a univariate analysis. A multivariate analysis of prognostic factors for AL amyloidosis revealed that GI involvement was not a significant predictor of OS (p = 0.447). CONCLUSIONS: The prognosis of patients with AL amyloidosis and GI involvement was poorer than those without GI involvement, and they presented with more organ involvement and more advanced disease than those without organ involvement.

Presence of autoantibody against ATTR Val30Met after sequential liver transplantation.

BACKGROUND: Recently, sequential liver transplantation has been performed with an explanted liver from a patient with familial amyloidotic polyneuropathy (FAP) because of the shortage of donors. However, metabolism of amyloidogenic transthyretin (ATTR), the pathogenic protein of FAP, has not been well studied in patients who have undergone sequential liver transplantation. The purpose of this study was to examine the changes in serum ATTR levels and to investigate the presence of an autoantibody in patients who underwent sequential liver transplantation with an explanted organ from a patient with heterozygotic FAP (FAP ATTR Val30Met). METHODS: This was a case study performed at the Kumamoto University School of Medicine, Kumamoto, Japan, and Kyoto University School of Medicine, Kyoto, Japan. Intervention occurred by sequential liver transplantation with an explanted FAP patient's liver. Levels of normal TTR and ATTR in the two patients who received the transplanted liver were analyzed by means of an enzyme-linked immunosorbent assay (ELISA) and a matrix-assisted laser desorption/time-of-flight mass spectrometry. In addition, the presence of an autoantibody against ATTR Val30Met was evaluated via ELISA using purified ATTR Val30Met from homozygotic FAP patients' sera. RESULTS: After the operation, the variant TTR levels were unexpectedly lower than levels of normal TTR in serum samples from patients with a transplanted liver from the FAP patient. An autoantibody against the variant TTR was detected on day 3 after the operation in the serum of those patients and continued to be present for at least 2 months after the operation. CONCLUSIONS: An autoantibody against the variant TTR may reduce the serum levels of variant TTR. Although the antibody may play a beneficial role in reducing the pathogenic protein, the long-term effect of the antibody must be investigated further.

Interleukin-1 signaling pathway as a therapeutic target in transthyretin amyloidosis.

INTRODUCTION: Inflammation is a key pathological hallmark of several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and familial amyloidotic polyneuropathy (FAP). Among all inflammatory cytokines associated with FAP, IL-1ß, in particular, has been implicated in playing a key pathogenic role. In the present study, we sought to investigate whether blocking IL-1ß signaling provides disease-modifying benefits in an FAP mouse model. METHODS: We assessed the effect of chronic administration of Anakinra, an IL-1 antagonist, on FAP pathogenesis in vivo, using real-time polymerase chain reaction (qPCR), semi-quantitative immunohistochemistry (SQ-IHC), western blot and nerve morphometric analyses. RESULTS: We found that treatment with Anakinra prevents transthyretin (TTR) extracellular deposition in sciatic nerve, protecting unmyelinated nerve fibers from aggregate-induced degeneration. Moreover, Anakinra administration significantly suppressed IL-1 signaling pathway and inhibited apoptosis and nitrative stress. CONCLUSIONS: The present work highlights the relevance of the IL-1 signaling pathway in the pathophysiology of FAP. Our results bring to light the importance of non-amyloid targets in the therapeutic strategies for this disorder. Thus, we propose the use of Anakinra as a potential therapeutic agent for TTR-related amyloidosis.

Impact of antibodies against amyloidogenic transthyretin (ATTR) on phenotypes of patients with familial amyloidotic polyneuropathy (FAP) ATTR Valine30Methionine.

BACKGROUND: This study investigated whether a relationship exists between the presence of de novo antibodies and the clinical manifestations of familial amyloidotic polyneuropathy (FAP). METHODS: Serum samples were collected from 25 Japanese and 6 Swedish FAP amyloidogenic transthyretin (ATTR) Valine30Methionine (V30M) patients, 4 asymptomatic Japanese ATTR V30M gene carriers, and 24 Japanese healthy volunteers. Study methods included enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. RESULTS: Three Japanese and 5 Swedish patients had significantly higher levels of antibodies against ATTR than did healthy volunteers and asymptomatic gene carriers (P<0.05). All 8 patients with higher antibody levels were late-onset cases. The ratio of wild-type TTR to ATTR V30M in serum from the high-antibody group was higher than that of the low-antibody group. ELISA results revealed two epitopes at positions 24-35 and 105-115 of ATTR V30M. We found a significant positive correlation between levels of the antibody at positions 24-35 and the age at FAP onset (r=0.751, P<0.05). An age-dependent increase in the occurrence of antibodies was observed in these patients with an epitope at positions 24-35. CONCLUSIONS: These findings may help explain the differences in early- and late-onset FAP and/or the progression of FAP.

Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant.

The mechanism of amyloid formation in familial amyloidotic polyneuropathy (FAP), a hereditary disorder associated with mutant transthyretin (TTR), is still unknown. It is generally believed that altered conformations exposing cryptic regions are intermediary steps in this mechanism. A TTR mutant--Y78F (transthyretin mutant with phenylalanine replacing tyrosine at position 78)--designed to destabilize the native structure has been shown to expose a cryptic epitope recognized by a monoclonal antibody that reacts only with highly amyloidogenic mutants presenting the amyloid fold or with amyloid fibrils. To test whether TTR deposition in FAP can be counteracted by antibodies for cryptic epitopes, we immunized with TTR Y78F, transgenic mice carrying the most common FAP-associated TTR mutant--V30M (transthyretin mutant with methionine replacing valine at position 30)--at selected ages that present normally with either nonfibrillar or TTR amyloid deposition. Compared to age-matched control nonimmunized mice, Y78F-immunized mice had a significant reduction in TTR deposition usually found in this strain, in particular in stomach and intestine; by contrast, animals immunized with V30M did not show differences in deposition in comparison with nonimmunized mice. Immunohistochemical analyses of tissues revealed that immunization with Y78F lead to infiltration by lymphocytes and macrophages at common deposition sites, but not in tissues such as liver, choroid plexus, and Langerhans islets, in which TTR is produced. These results suggest that Y78F induced production of an antibody that reacts specifically with deposits and leads to an immune response effective in removing/preventing TTR deposition. Therefore, TTR immunization with selected TTR mutants has potential application in immune therapy for FAP.