AA Amyloid Research Articles

Histopathological and Immunohistochemical Characteristics of Different Types of Cardiac Amyloidosis.

Cardiac involvement is the most important factor determining prognosis in patients with systemic amyloidosis. This retrospective observational study of 98 patients with amyloidosis was undertaken to assess the amyloid types that are most likely to affect the heart, describe histopathological and clinical features of cardiac amyloidosis, and estimate the number of cases not diagnosed clinically prior to death. All cases were divided into two groups based on the method of examination. The first group included 46 patients with cardiac amyloidosis revealed via endomyocardial biopsies (EMBs), and the second group included 52 amyloidosis patients who did not undergo EMBs, in whom cardiac involvement was identified only at autopsy. The EMBs demonstrated that AL amyloidosis was detected in 21 (46%) specimens, ATTR amyloid in 24 cases (52%), and AA amyloid in 1 case (2%). The autopsy reports defined 15 (46%) cases of AL amyloidosis, 21 (40%) of ATTR and 16 (31%) of AA amyloidosis. It should be noted that a clinical diagnosis of ATTR amyloidosis was made only in 9.5% of patients from the autopsy group, suggesting that ATTR may be an underdiagnosed cause of heart failure in elderly patients. The most intense amyloid deposits were determined in biopsy and autopsy specimens of patients with AL kappa amyloidosis, underlying a poorer prognosis.

ELISA-4-amyloid: diagnostic accuracy of an ELISA panel for typing the four main types of systemic amyloidosis in subcutaneous abdominal fat tissue samples

Background Reliable typing of amyloid is essential. Amyloid extraction from tissue enables immunochemical typing of the precursor protein using an enzyme-linked immunosorbent assay (ELISA). Objective To assess the diagnostic accuracy of a panel of ELISAs for typing the four main types (AA, ATTR, AL-kappa and AL-lambda amyloid). Methods From 1996 to 2023 subcutaneous abdominal fat tissue aspirates were obtained from 1339 amyloidosis patients and 868 controls. Amyloid was visually graded 0–4+ in Congo red-stained smears. Amyloid extracted from tissue by Guanidine was typed using a panel comprising four ELISAs. Results All amyloid protein concentrations in extracts correlated with amyloid grade in smears. Typing sensitivity was low (23.3%) in samples with grade 1+/2+ amyloid. Overall typing sensitivity of the panel was 81.6% for all easily visible amyloid (grade 3+/4+): high for AA (98.8%) and ATTR (96.8%) and fair for AL-kappa (66.7%) and AL-lambda (75.9). Overall typing specificity was 98.0% and the overall positive predictive value was 98.0%. Conclusions We describe a highly specific ELISA panel for routine typing of the main amyloid types in fat tissue. Until more sensitive typing techniques will become generally available, typing easily visible amyloid in fat tissue using this ELISA panel is reliable, affordable and straightforward.

#2698 Regression of amyloid deposits after treatment with tocilizumab

Abstract Background and Aims AA amyloidosis is a multisystem disease characterized by the deposition of serum amyloid A protein, which is secondary to chronic inflammation. Tocilizumab (an interleukin-6 inhibitor monoclonal antibody) is effective in suppressing inflammation, normalizing serum amyloid A protein levels and inducing remission in patients with amyloidosis. Recently, tocilizumab treatment has been associated with the disappearance of amyloid deposits. Results We report a case of AA amyloidosis secondary to chronic arthritis in a 61-year-old woman referred to our hospital in 2011 due to oligoarthritis of the knees and elevation of acute-phase reactants. Corticosteroids and methotrexate were prescribed, without clinical response. Two years later, the patient presented with foamy urine, nocturia, sweating and dizziness. An elevated C-reactive protein (CRP), erythrocyte sedimentation rate and nephrotic-range proteinuria were found. Rectal and oral mucosa biopsies were performed and amyloid AA deposits were detected in both (Fig. 1A, B). Magnetic resonance imaging (MRI) of the right knee showed arthropathy due to amyloid deposition (Fig. 2). Intravenous monthly tocilizumab was prescribed with rapid improvement of CRP, proteinuria (Fig. 3) and nephrotic syndrome symptoms. Arthritis also improved significantly. Two years later, a new biopsy of the rectal mucosa did not show amyloid deposits (Fig. 1C, D) and right knee MRI was normal, without evidence of amyloid synovitis (Fig. 4). Eight years later, the patient continues treatment with tocilizumab and remains clinically stable, with no evidence of recurrence. Literature review: The efficacy of tocilizumab has been described mainly in patients with rheumatoid arthritis, but also in AA amyloidosis secondary to systemic juvenile idiopathic arthritis, familial Mediterranean fever, psoriatic arthritis, ankylosing spondylitis, Still's disease, Muckle-Wells syndrome, Behçet syndrome, polyarteritis nodosa and Takayasu arteritis. Some of the studies report long-term effectiveness of up to 9 years. In 11 case reports of patients with AA amyloidosis treated with tocilizumab, a follow-up biopsy was performed and a reduction or disappearance of the deposits was also observed. Lane et al. analyzed pre- and on-treatment serial scintigraphy scans in 20 patients with AA amyloidosis, showing that AA amyloid deposits either regressed or did not increase whilst on tocilizumab. We have not identified any study demonstrating the disappearance of amyloid arthropathy in MR images. Conclusion Tocilizumab treatment controls chronic inflammatory disease and improves symptoms of AA amyloidosis. Long-term treatment with tocilizumab may remove amyloid deposits from tissues, leading to a definitive cure of this disease.

#2108 Secondary amyloidosis in a cancer patient on pembrolizumab—an unusual finding

Abstract Background and Aims With the widening horizon of ICPIs in the management of oncological disorders, we require to remain vigilant of the possible iRAEs. We present a case report of secondary amyloidosis in at metastatic non-small cell lung carcinoma (NSCLC) patient treated with Pembrolizumab. We aim to describe the clinic-biological & histopathological characteristics of ICPI-related kidney injury which may be beyond interstitial nephritis & its response to treatment & the role of kidney biopsy to define ICPI-related kidney injury. Treatment is challenging due to a paucity of relevant data, implicating further studies. Treatment with colchicine and steroids, tocilizumab in the setting of elevated interleukin-6 (IL6) levels have been reported in case series; unfortunately no appreciable renal benefit were noted. Method 77 year male, diabetic & hypertensive on regular medication for >15 yr, reformed smoker (≈50 pack-years of cigarette) diagnosed with NSCLC (EGFR–wild type, PDL1–70%), received Pembrolizumab (13 cycles) & Paclitaxel + Carboplatin & was in remission. Follow up PET showed partial response—new lesions in brain. Gemcitabine added to the treatment & had received 2 cycles, till hospitalization with febrile episode, decreased oral intake & progressive edema. Clinical evaluation was non-contributory. Hematology & biochemical evaluation (Table 1) showed raised serum creatinine 1.1 mg/dL (baseline 1 mg/dL), urinalysis—2+ protein, quantification showed 2.9295 g/24 h → 6.38 g/24 h (baseline uPCR-0.19 g/g). Abdominal ultrasound & ECHO finding were not conclusive. Fundoscopy showed no evidence of proliferative diabetic or hypertensive retinopathy. Results Kidney Biopsy (Figure) was done. LM showed, few of 8 gloms with mild segmental mesangial expansion due to amorphous extracellular deposits which was PAS & silver negative but showed congophilia (Fig. A,B,C,D). If negative for albumin, IgG, IgA, C3, C1q & had 1+ granularity in the mesangium on IgM staining & showed no light chain restriction in glomeruli & tubular cast for kappa & lambda. IHC (Fig. E) was positive for serum amyloid associated proteins. EM revealed minimal subendothelial & mesangial expansion due to extracellular material organized into randomly arranged fine fibrils, & no significant foot process effacement, electron dense deposits along the capillary basement membrane or within mesangium, glomerular basement membrane, thereby excluding possibility of light chain deposition disease. Conclusion Secondary amyloidosis with ICPI use has uncommonly & with Pembrolizumab rarely been reported. Treatment is challenging due to paucity of literature. Hassan Izzedine et al. in their case series, on Pembrolizumab treated cancer patient [N = 676], showed 12 patients (7 men) (Incidence 1.77%) developed AKI (n = 10) &/or Proteinuria (n = 2), median duration of use 9 months. All (n = 12) underwent renal biopsy, which showed AIN in 4, ATI in 5, MCD in 2. Recovery happened with withdrawal of drug coupled with corticosteroid. Reintroduction resulted in more severe recurrence of AIN in 1 patient only. Lapman et al. reported 1 case of secondary amyloidosis in cancer patients treated with Pembrolizumab. 42yr male Caucasian, had no reported chronic illness, except Asthma. Similarly developed significant proteinuria (Urine Pro 3+) & AKI (Sr. Creat 1.58 mg/dl; Baseline 1.0 mg/dl) after 90 days of Pembrolizumab therapy. Under went 2 Kidney Biopsy–1st Biopsy revealed AA amyloid. AA amyloidosis was attributed to Pembrolizumab as repeat Bx revealed progressive deposits despite remission of malignancy, but his kidney disease progressed, and had to be initiation on maintenance hemodialysis. Stopping Pembrolizumab & corticosteroids led to resolution of proteinuria in our patient too.

Preclinical evaluation of Tc-99m p5+14 peptide for SPECT detection of cardiac amyloidosis.

Amyloid deposition is a cause of restrictive cardiomyopathy. Patients who present with cardiac disease can be evaluated for transthyretin (TTR)-associated cardiac amyloidosis using nuclear imaging with 99mTc-labeled pyrophosphate (PYP); however, light chain-associated (AL) cardiac amyloid is generally not detected using this tracer. As an alternative, the amyloid-binding peptide p5+14 radiolabeled with iodine-124 has been shown to be an effective pan-amyloid radiotracer for PET/CT imaging. Here, a 99mTc-labeled form of p5+14 peptide has been prepared to facilitate SPECT/CT imaging of cardiac amyloidosis. A synthesis method suitable for clinical applications has been used to prepare 99mTc-labeled p5+14 and tested for peptide purity, product bioactivity, radiochemical purity and stability. The product was compared with99mTc-PYP for cardiac SPECT/CT imaging in a mouse model of AA amyloidosis and for reactivity with human tissue sections from AL and TTR patients. The 99mTc p5+14 tracer was produced with >95% yields in radiopurity and bioactivity with no purification steps required and retained over 95% peptide purity and >90% bioactivity for >3 h. In mice, the tracer detected hepatosplenic AA amyloid as well as heart deposits with uptake ~5 fold higher than 99mTc-PYP. 99mTc p5+14 effectively bound human amyloid deposits in the liver, kidney and both AL- and ATTR cardiac amyloid in tissue sections in which 99mTc-PYP binding was not detectable. 99mTc-p5+14 was prepared in minutes in >20 mCi doses with good performance in preclinical studies making it suitable for clinical SPECT/CT imaging of cardiac amyloidosis.

Early Development and Pre-Clinical Evaluation of a Fluorine-18 Labeled Peptide p5+14 for PET/CT Imaging of Cardiac Amyloidosis

Background:Systemic amyloidosis is a rare protein misfolding disorder, where patients can present with varied symptoms due to the diverse organ involvement. This is particularly true of patients with light chain associated (AL) amyloidosis, which makes early and accurate diagnosis challenging. Currently there are no FDA-approved methods that allow visualization of amyloidosis in patients. Nuclear imaging is a promising method for early detection of amyloid. We have developed a novel amyloid-reactive peptide, designated p5+14, capable of binding AL, as well as other types of amyloid, via multivalent electrostatic interactions. Peptide p5+14 has been successfully radiolabeled with radioiodine and technetium-99m and evaluated clinically, labeled with iodine-124, in a PET/CT imaging study of patients with amyloidosis (NCT03678259). While there are numerous advantages to using I-124 as the radionuclide for amyloid imaging, fluorine is a viable alternative for detecting cardiac amyloidosis. Fluorine-18 has a shorter half-life (108 min as compared to 4.2 days) thus reducing the internal radiation dose to patients. Moreover, F-18 is the most common nuclide used in PET imaging. We have previously generated a 18F-labeled variant of p5+14; however, the reaction was inefficient and not translatable. Here we present a novel development strategy for generating 18F labeled p5+14 using a commercially available precursor that can bind AL amyloid and perform initial characterization of the reagent in a murine model of inflammation-associated systemic AA amyloidosis. Methods: Isotopic exchange chemistry using a silicon-fluoride-acceptor (SiFA) method was used to label peptide p5+14 with F-18. The peptide precursor was purchased from Almac Life Sciences (Penicuik, UK). 18F was purchased from PetNet (Siemens Healthineers, Knoxville, TN) and prepared for reaction according to standard methods. After drying, F-18 (1000 - 1900 MBq ) was dissolved in anhydrous acetonitrile and the pH neutralized before the addition of peptide precursor. Radiolabeling was performed at room temperature for 5 min. After standard purification of the product, radiochemical purity studies demonstrated that it was stable, with no significant loss of F-18 for at least 5 hours. Radiofluorinated p5+14 was assessed in bioactivity “pulldown” assays to ensure binding to synthetic AL amyloid-like fibrils and human AL amyloid extracts. The radiolabeled peptide was also diluted in PBS and injected IV in the lateral tail vein of mice with severe systemic AA amyloidosis. After 1 h, mice were euthanized small animal PET/CT imaging performed and tissue biodistribution measurements were conducted using an automated Wizard 3 gamma counter (1480 Wallac Gamma Counter, Perkin-Elmer). Results: Using optimized synthesis conditions, the non-decay corrected radiochemical yield was 55±4.5%. Radiochemical purity was 99±1% at end of synthesis. Peptide purity was shown to be 93.7±4.9% by assessed using HPLC. Radiochemical stability was similarly evaluated using an HLPC method and remained >97.5% radiochemical purity at 5 hours after synthesis. The binding of 18F-p5+14 to synthetic AL amyloid like fibrils composed of a l6 variable domain was 96.2%, mimicking data using the radioiodinated peptide, and 87.2% to human AL amyloid extracts. Background binding to control beads was 17.4%. In mice, accumulation of 18F-peptide in the liver and spleen, the organs with most AA amyloid deposits in this mouse model, was readily evident in PET images taken 1 h post injection. There was also evidence of hepatic clearance of the peptide, manifest as a prominent gall bladder in the images. There was no accumulation of F-18 in bone, indicating positive in vivo stability. Imaging of amyloid-free wild type mice showed rapid clearance with radioactivity in the urinary bladder, gall bladder and gastrointestinal tract with little or no hepatosplenic radiotracer retention. Conclusion: Radiofluorination of peptides, such as p5+14, has been made more efficient through the use of novel precursors amenable to isotopic exchange. 18F-peptide binds amyloid effectively and may serve as a next generation radiotracer for the detection of cardiac amyloid by PET/CT imaging. Acknowledgments: EW is a doctoral student in the Comparative and Experimental Medicine Program. This study was supported by the Amyloidosis and Cancer Theranostics Program and the UTGSM.

AA Amyloid Masquerading as C3 Glomerulonephritis

AA Amyloid Masquerading as C3 Glomerulonephritis

Investigation of serum amyloid a within animal species focusing on the 1-25 amino acid region

AA amyloidosis, characterized by the misfolding of serum amyloid A (SAA) protein, is the most common amyloid protein disorder across multiple species. SAA is a positive-acute phase protein synthesized by the liver in response to inflammation or stress, and it normally associates with high-density lipoprotein at its N-terminus. In this study, we focused on the 1-25 amino acid (aa) region of the complete 104 aa SAA sequence to examine the aggregation propensity of AA amyloid. A library comprising eight peptides from different species was assembled for analysis. To access the aggregation propensity of each peptide region, a bioinformatic study was conducted using the algorithm TANGO. Congo red (CR) binding assays, Thioflavin T (ThT) assays, and transmission electron microscopy (TEM) were utilized to evaluate whether the synthesized peptides formed amyloid-like fibrils. All synthetic SAA 1-25 congeners resulted in amyloid-like fibrils formation (per CR and/or ThT staining and TEM detection) at the exception of the ferret SAA1-25 fragment, which generated plaque-like materials by TEM. Ten residues were preserved among SAA 1-25 congeners resulting in amyloid-like fibrils, i.e. F6, E9, A10, G13, D16, M17, A20, Y21, D23, and M24. Amino acid residues highlighted by this study may have a role in increasing the propensity for amyloid-like fibril formation. This study put an emphasis on region 1-25 in the mechanism of SAA1 misfolding.

AMYLOIDOSIS IN CAPTIVE EUROPEAN EASTERN BONGO (TRAGELAPHUS EURYCERUS ISAACI): PREVALENCE, PREDICTIVE FACTORS, ORGAN PREDILECTION, AND SERUM AMYLOID A CONCENTRATIONS.

Amyloidosis is frequently identified during postmortem examination of captive eastern bongo (Tragelaphus eurycerus isaaci) in the European Endangered Species Programme (EEP). However, its significance and etiopathogenesis are poorly understood. The objective of this study was to investigate the prevalence of amyloidosis within this population and identify potential predictive factors for the presence of disease. Postmortem reports obtained from 24 EEP institutions were analyzed and assessed for evidence of amyloidosis. Seventy-two individuals had histopathological assessment performed after gross postmortem examination and were included in the study. Further histopathological analysis was performed on Congo red-stained slides from 26 individuals, and organ predilection sites were identified. Immunohistochemical analysis was performed in six individuals to identify the type of amyloid present. Serum amyloid A (SAA) analysis was performed on blood samples from 34 individuals, and concentrations in affected and unaffected individuals were compared. Amyloidosis was reported in 26 animals (36%). The association between the presence of amyloidosis and sex, age, or body condition was not statistically significant. However, amyloidosis was not identified in any individuals under the age of 6 yr. The presence of chronic inflammatory conditions was the only statistically significant predictive factor for the presence of amyloidosis (P = 0.03). Chronic inflammatory conditions present included nephritis, enteritis, and pneumonia. The majority of affected animals presented with amyloid deposition in multiple organs, with the liver and kidneys being most commonly affected. Immunohistochemistry confirmed the presence of AA amyloid. The association between the presence of amyloidosis and SAA values measured on a single occasion was not statistically significant. This study identified a high prevalence of amyloidosis within the captive European eastern bongo population associated with chronic inflammatory conditions. Antemortem diagnosis of amyloidosis remains challenging, and this study indicates that SAA protein concentrations are not a reliable indicator for the presence of amyloidosis.

Rapid Clinical Improvement of Multicentric Castleman Disease (MCD) with Renal Involvement Following Treatment with Tocilizumab: AA Amyloidosis as a Possible Renal Involvement of MCD.

Castleman disease (CD) is a lymphoproliferative disorder that manifests as hypergammaglobulinemia and severe inflammation with multiorgan involvement. However, renal involvement has been infrequently described in CD. We present a case of a 63-year-old Japanese male patient with multicentric CD (MCD) in whom kidney involvement, including impaired renal function and massive proteinuria, is present. He had a 2-year history of inflammatory arthritis and was referred to our clinic with newly developed proteinuria, renal dysfunction, and elevated levels of acute-phase proteins. Abdominal computed tomography scan revealed hepatosplenomegaly, including mesenteric and inguinal lymph node enlargements. The patient underwent inguinal lymph node resection. Excisional biopsy of the inguinal lymph node showed multiple lymphoid follicles and expansion of interfollicular areas by marked plasmacytosis consistent with mixed type CD. The patient was diagnosed with human herpes virus 8-negative MCD according to the international diagnostic criteria for CD. Diagnostic renal biopsy was not performed following the medical viewpoint. Tocilizumab (TCZ) treatment was highly effective in reducing proteinuria and stabilizing renal function, as well as improving other clinical symptoms. The patient responded to TCZ treatment, and the renal involvement was rapidly improved. Our preliminary immunohistochemical analysis indicated AA amyloid deposits in urinary epithelial cells suggesting a possible renal involvement of AA amyloidosis. TCZ could potentially be one of the therapeutic options in patients with MCD with renal involvement.

Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-β amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. Prion-like transmission was reported as possible cause of extreme AA amyloidosis prevalence in captive animals, e.g. 70% in cheetah and 57–73% in domestic short hair (DSH) cats kept in zoos and shelters, respectively. Herein, we present the 3.3 Å cryo-EM structure of AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure, deceased in a shelter with extreme disease prevalence. The structure reveals a cross-β architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Inclusion of an eight-residue insert unique to feline SAA contributes to increased amyloid stability. The presented feline AA amyloid structure is fully compatible with the 99% identical amino acid sequence of amyloid fragments of captive cheetah.

A Case of ALECT2 Renal Amyloidosis Associated with IgG4 Related Kidney Disease, Membranous Nephropathy and Early Diabetic Kidney Injury

Abstract Introduction/Objective ALECT-2 amyloidosis is a rare type of amyloidosis that mostly involves kidneys with other organs rarely affected. It has a high prevalence among patients of Hispanic descent. Membranous nephropathy is one of the most common causes of proteinuria in adults. IgG4-related disease is a systemic disease, which commonly involves the pancreas, but occasionally affects the kidney and manifests as chronic renal insufficiency. Here we describe a very unusual case of concurrence of membranous nephropathy, IgG4 disease involving the kidney, ALECT2 amyloidosis, and early diabetic kidney injury. Methods/Case Report A 49-year-old Hispanic male patient with a history of diabetes and IgG4-related autoimmune pancreatitis and primary sclerosing cholangitis presented with abrupt onset of proteinuria and hypoalbuminemia. A kidney biopsy was performed and showed severe interstitial plasma cell-rich inflammatory infiltrates and interstitial fibrosis which had a storiform pattern. The glomerular basement membranes (GBM) showed focal pinpoint holes but no spikes by silver stain. Immunofluorescence microscopy (IF) showed diffuse and finely granular capillary loop staining for IgG, with Kappa and lambda light chains of equal intensity. IF for Anti-phospholipase A2 receptor (PLA2R) was negative. Immunohistochemical (IHC) stain showed IgG4 positivity in about 60% of IgG-positive plasma cells. Congo red was positive for birefringent deposits predominantly in the interstitium and arteriolar walls with focal deposits in the glomerular mesangium and capillary wall. IHC stain for Amyloid AA and DNAJB9 were negative. Electron microscopy showed scattered subepithelial immune complex-type electron dense deposits consistent with membranous nephropathy, randomly oriented fibrils in interstitium, mesangium and GBM, consistent with amyloidosis, and thickening of GBM (average 559 nm), consistent with early diabetic kidney change. The tissue was sent for mass spectrometry which showed a peptide profile consistent with ALECT-2 (Leukocyte chemotactic factor 2) type amyloidosis. Results (if a Case Study enter NA) NA Conclusion In up to a third of cases reported in the literature, a concomitant renal pathology was present. Diabetic nephropathy was the most common concurrent pathology, to be followed by IgA nephropathy and membranous nephropathy. However, the concurrence of membranous nephropathy, IgG4 disease involving the kidney, ALECT2 amyloidosis, and early diabetic kidney injury has never been described before.

Hepatic Amyloidosis in a Chronically Entangled Grey Seal (Halichoerus grypus)

Grey seal (Halichoerus grypus) entrapment in fishing gear is well documented, consisting of two forms: peracute underwater entrapment and chronic entanglement. We now highlight a previously undescribed sequela to chronic entanglement in a female grey seal estimated to be at least 2 years of age. The animal was first observed in September 2018 on the coast of north Cornwall, southwest England, with a large encircling neck wound consistent with monofilament net entanglement. In April 2021, it was admitted for attempted rehabilitation but had to be euthanized after 9 days due to clinical deterioration despite treatment. At post-mortem examination, the seal was in poor nutritional state, the nose to flipper length was low for its estimated age and the liver was markedly enlarged, pale and friable in texture with evidence of recent and previous hepatic haemorrhage. Histopathology revealed hepatic amyloidosis and evidence of amyloid in one kidney and one adrenal gland. Proteomic analysis of microdissected amyloid from the liver indicated type AA amyloid. Chronic entanglement is the most plausible cause of AA amyloidosis in this animal, indicating that amyloidosis should be considered as a pathological sequela and welfare concern associated with chronic entanglement of grey seals.

N-terminal peptide fragment constitutes core of amyloid deposition of serum amyloid A: An imaging mass spectrometry study.

Serum amyloid A (SAA) is an acute phase protein, which undergoes structural changes and deposits in the extracellular matrix, causing organ damage. Systemic AA amyloidosis is a relatively common amyloid subtype among the more than 30 amyloid subtypes, but the mechanism of amyloid fibril formation remains unclear. In this study, we investigated the tissue distribution of SAA derived peptides in formalin-fixed paraffin embedded (FFPE) specimens of human myocardium with amyloidosis using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). In the whole SAA protein, four trypsin-digested peptides in the range of SAA2-67 were visualized and the N-terminal peptide; SAA2-15, was selectively localized in the Congo red-positive region. The C-terminal peptides; SAA47-62, SAA48-62, and SAA63-67 were detected not only in the Congo red-positive region but also in the surrounding negative region. Our results demonstrate that the N-terminal SAA2-15 plays a critical role in the formation of AA amyloid fibril, as previously reported. Roles of the C-terminal peptides require further investigation.

Detection and Typing of Renal Amyloidosis by Fluorescence Spectroscopy Using the Environmentally Sensitive Fluorophore K114.

To demonstrate that spectral analysis using the K114 fluorophore can detect and differentiate AL and AA renal amyloidosis. Kidney biopsies from patients with AL amyloidosis, AA amyloidosis, and normal samples with no evident pathology were stained with Congo Red and K114. The specimens were imaged on a spectral confocal microscope. Congo Red displayed homogeneous spectra across the three tissue types while K114 chromatically distinguished between normal tissue, AL amyloid, and AA amyloid. Additionally, Congo Red displayed an increased risk of false positive staining compared to K114. Spectral phasors computed from K114-stained tissue sections quantitatively differentiated the three tissue types. K114-stained amyloid deposits displayed a significantly greater increase in brightness after 50 images acquired in rapid succession compared to normal tissue. Quantitative analysis of intensity changes in the background of diseased tissue also differentiated AL and AA amyloid samples, suggesting widespread amyloid deposition. Both amyloid and the backgrounds of diseased samples red-shifted while normal tissue blue-shifted in response to repeated imaging, supporting this theory. K114 staining of renal biopsies is a promising technique to detect and differentiate types of renal amyloidosis. Due to the advantages this method has over traditional Congo Red staining, the techniques presented here warrant further development for potential use in clinical settings.

Morphological pattern of non-diabetic nephropathy in type 2 diabetes mellitus patients

Abstract. The aim of this study was to evaluate the contribution of kidney biopsy performed with an appropriate indication to diagnosis and treatment in diabetic patients with nephropathy.
 Methods: In this retrospective study 32 type 2 diabetes patients who underwent kidney biopsy in a single center between 2012-2019 were included. Kidney biopsy indications were determined as patients with diabetes without diabetic retinopathy and with proteinuria above 1 g/day.
 Results: Diabetic (DN) and non-diabetic (NDN) nephropathies were diagnosed with renal biopsy. In 14 (43.7%) of 32 patients, NDN was reported in histopathological evaluation. Membranous nephropathy was detected in 4 of these patients, focal segmental glomerulosclerosis (FSGS) in the other 4 patients, light chain disease in 2 patients, IgA nephropathy in 2 of the patients, minimal change nephropathy in another patient, and finally AA amyloid in one patient. NDRD seen superimposed on DN (DN + interstitial nephritis and DN + FSGS) was observed in 2 patients. DN was detected in 16 (50%) of 32 type 2 diabetic patients.
 Conclusion: Kidney biopsy in patients with type 2 diabetes is an important tool for diagnosing NDN, choosing the right treatment tactics and determining kidney prognosis.

Revisiting misfolding propensity of serum amyloid A1: Special focus on the signal peptide region

AA amyloidosis is the result of overproduction and aberrant processing of acute-phase serum amyloid A1 (SAA1) by hepatocytes. Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein undergoes a cleavage of 18 residues consisting of the signal peptide at the N-terminal region. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region. We first examined the signal peptide amino acid SAA derived from different animal species. A library of 16 peptides was designed to evaluate the propensity of aggregation. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using in silico Tango program, thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and seeding with misfolded human SAA1 signal peptide. After 7 days of incubation, most of the SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 100 μM in 50 mM Tris buffer at 37 °C by TEM. All peptides were able to generate fibrils at a higher concentration, i.e 500 μM in 25 mM Tris buffer with 50% HFIP, by ThT. All SAA1 signal synthetic peptides designed from the different animal species had the propensity to misfold and form fibrils, particularly in species with low occurrence of systemic amyloidosis. The human SAA1 signal peptide region was capable to seed the SAA1 1–25 and 32–47 peptide regions. Characterizing fibrillar conformations are relevant for seeding intact and/or fragmented SAA, which may contribute, to the mechanism of protein misfolding. This research signifies the importance of the signal peptide region and its possible contribution to the misfolding of aggregation-prone proteins.

Misfolding Propensity of SAA1 Fragments: Special Focus on the Signal Peptide Region

AA amyloid is the most common form of systemic amyloidosis reported in human medicine and across animal species. AA amyloidosis is the result of over‐production by hepatocytes and aberrant processing of acute‐phase serum amyloid A1 (SAA1). Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein is of 122 amino acids (aa) and undergoes a first cleavage of 18 residues consisting of the signal peptide at the N‐terminal region. The aggregation propensity of the signal peptide region has never been studied. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region considering that SAA1 is going through further cleavage processes. The goal of this research was to determine the propensity of the SAA1 signal peptide region to generate fibrils. We examined the signal peptide amino acid sequence of serum amyloid A1 (SAA1) derived from different animal species. A library of the 25 peptides was designed to study the fibril formation using in silico analysis and in vitro assays. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using thioflavin T fluorescence assays, Congo red binding assays, and transmission electron microscopy. After 7 days of incubation, 22 SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 500 µM with 50% hexafluoroisopropanol at 37 ºC. The signal peptide region might be important in contributing to the misfolding of aggregation‐prone proteins. Characterizing these conformations are relevant as they may be involved in seeding intact and/or fragmented SAA1, contributing to mechanism of misfolding. This information could be important for continuation studies in the understanding of misfolding disorders.

Prognostic value of histopathological scoring and grading in patients with renal AA amyloidosis.

The amount and distribution pattern of amyloid deposits may contribute to renal function and outcome, given the great diversity of renal involvement in amyloidosis. The aim of this study was to analyze the impact of histological characteristics of patients with biopsy-proven renal AA amyloidosis (AAA)on renal outcome. Renal biopsies of 37 patients with AAA were re-evaluated. The distribution pattern of glomerular amyloid (GA) deposits was classified, the extent of amyloid deposits in glomeruli, vessel, and interstitium and other histopathologic lesions were scored, and renal amyloid prognostic score (RAPS) was determined by summing all scores. Their potential prognostic relevance on renal outcome was investigated. GA and vascular amyloid (VA) deposits were noted in all cases, interstitial amyloid (IA) was detected in 70.2%. GA deposits were predominantly seen in diffuse mesengiocapillary fashions (class IV) (51.4%). GA class, the extent of GA, VA, IA deposit, and RAPS, as well as interstitial fibrosis (IF) and interstitial inflammation were correlated to renal function at diagnosis. During the median follow-up of 52months, 13 patients developed doubling of serum creatinine or end stage renal disease and they had a higher degree of GA and VA load (p = 0.03 and p = 0.042, respectively) as compared to the remaining patients. VA load, but not GA and RAPS grade, was associated with poor renal outcome (HR 3.016, 95% CI 1.45-6.25, p = 0.003). Baseline renal function is closely linked to the extent of AA amyloid deposit in renal parenchyma but only VA load was a predictor of renal outcome in AAA patients.

Rare cause of thyroid enlargement: Localized AA amyloid goiter - A case report.

IntroductionAmyloidosis is extracellular deposition of fibrillary amyloid proteins in various organs. Amyloid infiltration in thyroid is common; however, the occurrence of clinically enlarged thyroid, subsequently leading to goiter, is a rare phenomenon.Case presentation36 years old female presented to our OPD with multinodular goiter. She subsequently underwent total thyroidectomy. Thyroidectomy specimen revealed amyloid deposition with characteristic congophilia and birefringence. Further evaluation revealed it to be AA amyloidosis. There were no features of systemic amyloidosis.DiscussionAmyloidosis is classified on the type of amyloid protein. Treatment of individual types of amyloidosis is diverse, and hence identification of the protein subtype is paramount.ConclusionAA amyloidosis localized primarily to thyroid is infrequent, as it usually occurs with chronic inflammatory conditions or infections. Currently, there are no guidelines for the treatment of localized AA amyloid goiter. We have evidence demonstrating the successful treatment of such a condition with no recurrence noted.