Regionalization of cell types in silk glands of Larinioides sclopetarius suggest that spider silk fibers are complex layered structures.

In order to produce artificial silk fibers with properties that match the native spider silk we likely need to closely mimic the spinning process as well as fiber architecture and composition. To increase our understanding of the structure and function of the different silk glands of the orb weaver Larinioides sclopetarius, we used resin sections for detailed morphology, paraffin embedded sections for a variety of different histological stainings, and a histochemical method for localization of carbonic anhydrase activity. Our results show that all silk glands, except the tubuliform glands, are composed of two or more columnar epithelial cell types, some of which have not been described previously. We observed distinct regionalization of the cell types indicating sequential addition of secretory products during silk formation. This means that the major ampullate, minor ampullate, aciniform type II, and piriform silk fibers most likely are layered and that each layer has a specific composition. Furthermore, a substance that stains positive for polysaccharides may be added to the silk in all glands except in the type I aciniform glands. Active carbonic anhydrase was found in all silk glands and/or ducts except in the type I aciniform and tubuliform glands, with the strongest staining in aggregate glands and their ductal nodules. Carbonic anhydrase plays an important role in the generation of a pH gradient in the major ampullate glands, and our results suggest that some other glands may also harbor pH gradients.

Tissue-based diagnosis of systemic amyloidosis: Experience of the informal diagnostic center at Uppsala University Hospital.

Diagnosis of systemic amyloidosis is a clinical challenge and usually relies on a tissue biopsy. We have developed diagnostic methods based on the presence of amyloid deposits in abdominal subcutaneous fat tissue. This tissue is also used to determine the biochemical type of amyloidosis, performed by western blot and immunohistochemical analyses with the aid of in-house developed rabbit antisera and mouse monoclonal antibodies. Mass spectrometric methods are under development for selected cases. The diagnostic outcome for 2018-2020 was studied. During this period, we obtained 1,562 biopsies, of which 1,397 were unfixed subcutaneous fat tissue with varying degrees of suspicion of systemic amyloidosis. Of these, 440 contained amyloid deposits. The biochemical nature of the amyloid was determined by western blot analysis in 319 specimens and by immunohistochemistry in further 51 cases.

Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee.

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.

Cardiac microcalcifications in transthyretin (ATTR) amyloidosis.

BACKGROUND: Bone tracers bind to amyloid-containing heart of most patients with ATTR amyloidosis. Amyloid deposits outside the heart are often scarce and bone scintigraphy is increasingly often used to diagnose cardiac involvement. However, the nature of the binding of bone tracers to the heart is not clear. OBJECTIVE: To identify possible calcium deposits in hearts with amyloid, explaining bone tracer binding. METHODS AND RESULTS: Formalin-fixed and paraffin embedded cardiac specimens from three patients with ATTR and one with AL amyloidosis, all with cardiac deposits, were studied. The specimens covered large parts of the heart. Sections were stained immunohistochemically for ATTR deposits and according to von Kóssa for calcifications. The study identified in all hearts, but particularly in the ATTR materials, focal, tight swarms of tiny calcifications. These were sometimes associated with amyloid but found as frequent in areas without such deposits. Autoradiography with [99mTc]Tc labelled 3,3-disphos-phono-1,2-propanodicarboxylic acid (DPD) revealed labelling in von Kóssa positive areas. Electron microscopically the particles were not amorphous but had a complex structured appearance and were often surrounded by a membrane, indicating a cellular origin. Labelling with antibodies against ubiquitin and P62 pointed to result from autophagy. CONCLUSIONS: Our study indicates that binding of skeletal probes to amyloid-containing hearts depends on an irregular presence of clouds of very tiny calcifications, which seem not to be directly associated with amyloid fibrils. Therefore, [99mTc]Tc-DPD bone scans can be considered surrogate markers of ATTR amyloid but have to be used carefully to estimate amyloid amount or disease progression.

Tissue biopsy for the diagnosis of amyloidosis: experience from some centres.

A reliable diagnosis of amyloidosis is usually based on a tissue biopsy. With increasing options for specific treatments of the different amyloid diseases, an exact and valid diagnosis including determination of the biochemical fibril nature is imperative. Biopsy sites as well as amyloid typing principles vary and this paper describes methods employed at some laboratories specialised in amyloidosis in Europe, Japan and USA.

AA amyloid in human food chain is a possible biohazard.

AA amyloidosis can be transmitted experimentally in several mammalian and avian species as well as spontaneously between captive animals, even by oral intake of amyloid seeds. Amyloid seeding can cross species boundaries, and fibrils of one kind of amyloid protein may also seed other types. Here we show that meat from Swedish and Italian cattle for consumption by humans often contains AA amyloid and that bovine AA fibrils efficiently cross-seed human amyloid ß peptide, associated with Alzheimer's disease.

Clinical ApoA-IV amyloid is associated with fibrillogenic signal sequence.

Apolipoprotein A-IV amyloidosis is an uncommon form of the disease normally resulting in renal and cardiac dysfunction. ApoA-IV amyloidosis was identified in 16 patients attending the National Amyloidosis Centre and in eight clinical samples received for histology review. Unexpectedly, proteomics identified the presence of ApoA-IV signal sequence residues (p.18-43 to p.20-43) in 16/24 trypsin-digested amyloid deposits but in only 1/266 non-ApoA-IV amyloid samples examined. These additional signal residues were also detected in the cardiac sample from the Swedish patient in which ApoA-IV amyloid was first described, and in plasma from a single cardiac ApoA-IV amyloidosis patient. The most common signal-containing peptide observed in ApoA-IV amyloid, p.20-43, and to a far lesser extent the N-terminal peptide, p.21-43, were fibrillogenic in vitro at physiological pH, generating Congo red-positive fibrils. The addition of a single signal-derived alanine residue to the N-terminus has resulted in markedly increased fibrillogenesis. If this effect translates to the mature circulating protein in vivo, then the presence of signal may result in preferential deposition as amyloid, perhaps acting as seed for the main circulating native form of the protein; it may also influence other ApoA-IV-associated pathologies. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Proteostasis of Islet Amyloid Polypeptide: A Molecular Perspective of Risk Factors and Protective Strategies for Type II Diabetes.

The possible link between hIAPP accumulation and ß-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, ß-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.

Abdominal fat pad biopsies exhibit good diagnostic accuracy in patients with suspected transthyretin amyloidosis.

BACKGROUND: The diagnostic accuracy of histopathological detection of transthyretin amyloid (ATTR) by Congo red staining of abdominal fat samples has been questioned since low sensitivity has been reported, especially for patients with ATTR cardiomyopathy. However, the outcome of surgically obtained fat pad biopsies has not yet been evaluated. The aim was to evaluate the diagnostic accuracy of skin punch biopsies from abdominal fat in patients with suspected ATTR amyloidosis. MATERIAL AND METHODS: Data were evaluated from patients who had undergone abdominal fat pad biopsies using a skin punch due to suspected amyloidosis from 2006 to 2015. The biopsies had been analysed using Congo red staining to determine the presence of amyloid, and immunohistochemistry or Western blot to determine the type of amyloidosis. The final diagnosis was based on the clinical picture, biopsy results and DNA sequencing. Minimum follow-up after the initial biopsy was 3 years. RESULTS: Two hundred seventy-four patients (61% males) were identified, and in 132 (48%), a final diagnosis of amyloidosis had been settled. The majority (93%) had been diagnosed with hereditary transthyretin (ATTRv) amyloidosis, and therefore subsequent analyses were focused on these patients. Overall, our data showed a test specificity of 99% and a sensitivity of 91%. Ninety-eight (94%) of the patients had neuropathic symptoms at diagnosis, whereas 57 (55%) had signs of amyloid cardiomyopathy. Subgroup analyses showed that patients with merely neuropathic symptoms displayed the highest test sensitivity of 91%, whereas patients with pure cardiomyopathy displayed the lowest sensitivity of 83%. However, no significant differences in sensitivity were found between patients with or without cardiomyopathy or between the sexes. CONCLUSIONS: Abdominal fat pad biopsies exhibit good diagnostic accuracy in patients with suspect ATTRv amyloidosis, including patients presenting with cardiomyopathy. In addition, the method enables typing not only of the precursor protein but also of the amyloid fibril type, which is related to the phenotype and to the outcome of the disease.

Amyloid nomenclature 2020: update and recommendations by the International Society of Amyloidosis (ISA) nomenclature committee.

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.

[Amyloid from insulin treatment: a pitfall for the pathologist and the diabetologist]. / Subkutant injicerat insulin kan ge amyloidinlagring - Diagnostiska dilemman och fallgropar för patologen och oanade konsekvenser för diabetologen.

Biopsies from six diabetic patients with subcutaneous amyloid deposits formed by injected insulin have been sent to our laboratory during the last year. In all but one of the six patients a subcutaneous adipose tissue biopsy was taken due to suspicion of systemic amyloidosis. Four of these patients had renal insufficiency, with monoclonal gammopathy of undetermined significance (MGUS) in three while the fifth had heredity for transthyretin amyloidosis. In the sixth patient a biopsy was taken due to subcutaneous nodules at insulin injection sites. In all biopsies, large amounts of amyloid were present and their biochemical nature was elucidated by immunohistochemistry or western blot. The risk of incorrect interpretation with misdiagnosis of systemic amyloidosis is underlined. The consequences this may have on insulin treatment are insufficiently studied.

Cryo-EM structure of a transthyretin-derived amyloid fibril from a patient with hereditary ATTR amyloidosis.

ATTR amyloidosis is one of the worldwide most abundant forms of systemic amyloidosis. The disease is caused by the misfolding of transthyretin protein and the formation of amyloid deposits at different sites within the body. Here, we present a 2.97 Å cryo electron microscopy structure of a fibril purified from the tissue of a patient with hereditary Val30Met ATTR amyloidosis. The fibril consists of a single protofilament that is formed from an N-terminal and a C-terminal fragment of transthyretin. Our structure provides insights into the mechanism of misfolding and implies the formation of an early fibril state from unfolded transthyretin molecules, which upon proteolysis converts into mature ATTR amyloid fibrils.

Transthyretin Glu54Leu - an unknown mutation within the Swedish population associated with amyloid cardiomyopathy and a unique fibril type.

For the first time, we report of a Swedish family of five individuals with a TTR Glu54Leu (p. Glu74Leu) mutation in the transthyretin gene. This mutation has been previously described a few times in the literature, but no phenotypic or clinical description has been done before. The most common mutation in the Swedish population is TTRVal30Met and is mostly found in the Northern part of Sweden. Interestingly, the TTRGlu54Leu mutation was found in the same endemic area. The main phenotype of the TTR Glu54Leu patients is severe cardiomyopathy, which resulted in heart transplantation for the index person. As previously seen for ATTR amyloidosis patients with mainly cardiomyopathy, the amyloid fibrils consisted of a mixture of full-length and fragmented TTR species. However, western blot analyses detected a previously unrecognized band, indicating that these patients may have a third, so far unrecognized, fibril composition type that is distinct from the usual type A band pattern.

Cryo-EM fibril structures from systemic AA amyloidosis reveal the species complementarity of pathological amyloids.

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 Å and 2.7 Å for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-ß sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar ß-arch conformations within the N-terminal ~21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs.