Amyloid-containing Tissues Research Articles

Supramolecular arrangements in human amyloid tissues using SAXS

Amyloid diseases are characterized by the accumulation of misfolded protein aggregates in human tissues, pose significant challenges for both diagnosis and treatment. Protein aggregations known as amyloids are linked to several neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis. The key goal of this research is to employ Small-Angle X-ray Scattering (SAXS) to examine the supramolecular structures of amyloid aggregates in human tissues. We present the structural analysis of amyloid using SAXS, which is employed directly to analyze thin tissue samples without damaging the tissues. This technique provides size and shape information of fibrils, which can be used to generate low-resolution 2D models. The present study investigates the structural changes in amyloid fibril axial d-spacing and scattering intensity in different human tissues, including kidney, heart, thyroid, and others, while also accounting for the presence of triglycerides in these tissues. Tissue structural components were examined at momentum transfer values between q = 0.2 nm−1 and 1.5 nm−1. The d-spacing is a critical parameter in SAXS that provides information about the periodic distances between structures within a sample. From the supramolecular SAXS patterns, the axial d-spacing of fibrils in amyloid tissues is prominent and exists within the 3rd to 10th order, compared to that of healthy tissues which do not have notable peak orders. The axial period of fibrils in amyloid tissues is within the scattering vector range 57.40–64.64 nm−1 while in normal tissues the range is between 60.68 and 61.41 nm−1, which is 3.0 nm−1 smaller than amyloid-containing tissues. Differences in d-spacing are often correlate with distinct pathological mechanisms or stages of disease progression. The application of SAXS to investigate amyloid structures in human tissues has enormous potential to further knowledge of amyloid disorders. This work will open the path for novel diagnostic instruments and therapeutic strategies meant to reduce the burden of amyloid-related diseases by offering a thorough structural examination of amyloid aggregates.

EDXRF and the relative presence of K, Ca, Fe and as in amyloidogenic tissues

Trace and minor elements play crucial roles in a variety of biological processes, including amyloid fibrils formation. Mechanisms include activation or inhibition of enzymatic reactions, competition between elements and metal proteins for binding positions, also changes to the permeability of cellular membranes. These may influence carcinogenic processes, with trace and minor element concentrations in normal and amyloid tissues potentially aiding in cancer diagnosis and etiology. With the analytical capability of the spectroscopic technique X-ray fluorescence (XRF), this can be used to detect and quantify the presence of elements in amyloid characterization, two of the trace elements known to be associated with amyloid fibrils. In present work, involving samples from a total of 22 subjects, samples of normal and amyloid-containing tissues of heart, kidney, thyroid, and other tissue organs were obtained, analyzed via energy-dispersive X-ray fluorescence (EDXRF). The elemental distribution of potassium (K), calcium (Ca), arsenic (As), and iron (Fe) was examined in both normal and amyloidogenic tissues using perpetual thin slices. In amyloidogenic tissues the levels of K, Ca, and Fe were found to be less than in corresponding normal tissues. Moreover, the presence of As was only observed in amyloidogenic samples; in a few cases in which there was an absence of As, amyloid samples were found to contain Fe. Analysis of arsenic in amyloid plaques has previously been difficult, often producing contradictory results. Using the present EDXRF facility we could distinguish between amyloidogenic and normal samples, with potential correlations in respect of the presence or concentration of specific elements.

Lung adenocarcinoma expressing receptor for advanced glycation end-products with primary systemic AL amyloidosis: a case report and literature review

BackgroundReceptor for advanced glycation end-products (RAGE), a receptor for amyloids, is constitutively expressed in lungs and generally observed to be downregulated in lung cancer tissues. However, increasing levels of RAGE or serum amyloids is associated with poor outcome in lung cancer patients. We report a rare case of primary systemic amyloid light-chain (AL) amyloidosis in biopsy-proven multiple organs with early-stage non-small cell lung cancer (NSCLC) that displayed strong staining for RAGE in the tumour tissue. Interestingly, compared with randomly selected lung cancer biopsy samples, including all representative histological subtypes of NSCLC and small-cell lung cancer, only the NSCLC in the present case showed strong expression for RAGE that can bind amyloids.Case presentationA 71-year-old woman was admitted to our hospital for comprehensive investigation of nephrotic syndrome. Computed tomography showed a small nodule in the right upper lung lobe with hilar mediastinal lymph node enlargement. Pathological examination of transbronchial biopsy samples of the nodule yielded a diagnosis of lung adenocarcinoma. Furthermore, the pathological detection of amyloid deposition in biopsy samples of a subcarinal lymph node, gastric and duodenal mucosa, cardiac muscle, and bone marrow led to a diagnosis of primary systemic AL amyloidosis with nephrotic syndrome and cardiomyopathy. In addition, RAGE was detected in lung tumour tissues surrounded by normal lung tissues with amyloid deposition.ConclusionThe RAGE positivity of the lung cancer cells in this case suggests an interaction between amyloid-containing tissues and RAGE-expressing cancer cells. Lung adenocarcinoma with RAGE expression may be a complication of underlying amyloidosis.

Probing the use of fluorescence spectroscopy as a novel diagnostic tool in patients with rheumatoid arthritis: Applicability in the detection of secondary amyloidosis

SummaryBackgroundSecondary amyloidosis is a frequently reported complication of rheumatoid arthritis. Currently, accepted diagnostic protocols for secondary amyloidosis involve histopathological and histochemical examinations of collected tissue specimens. The purpose of the current report was to evaluate the value of fluorescence spectroscopy as a supplementary tool in the diagnosis of secondary amyloidosis.Material/MethodsTissue specimens were collected from abdominal folds, gingiva or rectal mucosa of 99 patients affected with rheumatoid arthritis. Tissue samples were subjected to preliminary clinical observations, histopathological examinations and laboratory tests. These procedures were used to subdivide tissue samples into either amyloid-containing or amyloid-free control subgroups. All collected tissue samples were examined with the use of a designated spectrofluorometer and fluorescence spectral images were generated.ResultsIt was found that fluorescence spectra for amyloid-containing tissues were typically characterized by a double emittance peak. In contrast, amyloid-free samples were characterized by fluorescence spectra with a single λmax value. Specimen collection site, age and sex did not appear to influence the morphology of electromagnetic spectra, which were generated for both amyloid-containing and amyloid-free tissue samples. The sensitivity of the fluorometric approach was ~78% and the specificity was 100%. Possible shortcomings of the technique may be due to the limit of detection of the instrument used.ConclusionsFluorescence spectroscopy may potentially be used as an effective, instantaneous and low-cost diagnostic tool for suspected secondary amyloidosis in patients affected with rheumatoid arthritis.

PTAA and B10: new approaches to amyloid detection in tissue—evaluation of amyloid detection in tissue with a conjugated polyelectrolyte and a fibril-specific antibody fragment

Aims: We analysed the suitability of two little known substances for the detection of amyloid in surgical pathology specimens, that is the conjugated polyelectrolyte polythiophene acetic acid (PTAA) and the camelid antibody domain B10.Methods: We compared the amyloid detection of PTAA and B10 to Congo red in 106 amyloid-containing tissue biopsies of diverse anatomical and precursor origin by evaluating the accordance in four grades (grade 0: no staining, grade 1: staining of <33% of the amyloid deposits, grade 2: 33–66% and grade 3: >66%).Results: PTAA showed grade 2–3 staining in 57 (54%) cases, while B10 presented this accordance in only 25 (24%) tissue biopsies. Grade 1 staining was found in 11 (10%) samples with PTAA and in 62 (58%) cases with B10. No staining at all (grade 0) occurred in 38 (36%) biopsies when using PTAA and in 19 (18%) cases when using B10.Conclusion: Although conformation-sensitive detection seemed promising, PTAA and B10 stain only a fraction of the examined amyloid samples when using routine surgical pathology settings. This study emphasises the necessity of having optimised pre-analytical protocols for recovery, storage and handling of samples if these novel amyloid ligands are to be used in routine diagnosis of amyloid.

A novel type of familial transthyretin amyloidosis, ATTR Asn124Ser, with co-localization of κ light chains

A 68-year-old Italian woman who had a clinical history of thyroidectomy in 2002 presented with slowly progressing renal insuffiency and non-nephrotic proteinurea in 2004. A renal biopsy showed the occurrence of amyloid; the thyroid biopsy previously taken also revealed amyloid infiltration. Other amyloid-containing tissues included bone marrow and heart. The plasma cell level in the bone marrow was found to be less than 5% and both serum and urine samples were positive for a monoclonal κ light chain band. DNA analysis unexpectedly revealed the presence of a novel transthyretin (TTR) mutation, ATTR Asn124Ser. Histologically, amyloid deposits in the thyroid had a homogeneous appearance with moderate Congophilia. In immunohistochemistry, a κ light chain antiserum showed positive immunoreactivity with amyloid deposits in the thyroid. Furthermore, a TTR antiserum, anti-TTR50-127, also recognized a number of amyloid deposits stained positive with the κ light chain antiserum. Overall, the κ light chain antiserum reacted with most of the amyloid deposits in the thyroid, whereas TTR immunoreactivity was scarcer, with a scattered appearance. In contrast, only the anti-TTR50-127 antiserum labeled amyloid in the kidney, albeit not all deposits. In this study, we report a patient having a novel TTR variant, ATTR Asn124Ser, with co-localization of κ light chains in the amyloid deposits in the thyroid tissue.

Influence of tissue fixation on the microextraction and identification of amyloid proteins

In surgical pathology, correct immunohistochemical identification of AL amyloidosis poses a particular problem. Immunostaining for lambda- or kappa-light chains is commonly encountered even in non-immunoglobulin-derived amyloidoses, which leads to a false-positive classification as AL amyloidosis. In this respect, microextraction of amyloid proteins from surgical pathology specimens and their subsequent biochemical characterization may prove useful in reaching the correct diagnosis. In this study, we investigated systematically the influence of fixation on the extraction of amyloid proteins from amyloid-containing tissue samples. Tissue samples were obtained from a patient with generalized AA amyloidosis and from a second patient with generalized AL amyloidosis. The samples were stored either unfixed or fixed in phosphate buffered 4% p-formaldehyde, methacarn, or Bouin for 3 days, 1 week, or 1 month. Thereafter, proteins were extracted according to the procedure of Layfield et al, separated by SDS-PAGE and subjected to Western blotting, using antibodies directed against AA amyloid and immunoglobulin-derived lambda-light chain. Following this procedure, a variety of differently sized AA amyloid or lambda-light chain immunoreactive protein bands were found in both patients, which is typical for amyloid proteins. Fixation time did not per se prohibit the extraction of these amyloid proteins from tissue samples, which remained detectable irrespective of fixation time. Although all three fixatives impaired the resolution of some, but not all, individual amyloid proteins, this procedure may help to confirm or reject a diagnosis of AL amyloidosis, because detection of several lambda- or kappa-light chain immunoreactive protein bands in the low-molecular-weight range (<20 kDa) is a common characteristic of their amyloid nature.

Acceleration of murine amyloidosis by implantation of amyloid-containing grafts

We examined the transmissibility of amyloidosis by the implantation of amyloid-containing tissue. If the transmissibility similar to prion diseases is applicable, using amyloid-containing tissue for transplantation in humans might be a risk factor. In this study, AA amyloidosis occurred in mice that underwent implantation of AA amyloid-containing grafts to the liver and subsequent inflammatory stimulation. AApoAII amyloidosis occurred after implantation of AApoAII amyloid-containing grafts to the liver or to the subcutaneous space without inflammatory stimulation. Both types of amyloidoses occurred in the recipient mice sooner than expected. Moreover, AA and AApoAII amyloid deposits were found at 12 weeks after implantation in mice given AApoAII amyloid-containing grafts and inflammatory stimulation. These results suggest that implanted amyloid deposits have an AEF effect and that implanted amyloid-containing tissue can promote and accelerate a different type of amyloidosis. In another experiment, mice received amyloid-containing or normal tissue grafts. The degree of amyloid deposition was compared after 6 days and 5 weeks of inflammatory stimulation and when the mice were killed. There was no obvious difference in the degree of amyloid deposition between each group, indicating that the lag-time is shortened by implantation of amyloid-containing tissue, resulting in severe amyloidosis in the short term.

Detection of serum amyloid A-derived proteins in formalin-fixed paraffin-embedded tissues: reliability of the method and expansion of its spectrum.

To further assess the reliability of the Shtrasburg method for detection of amyloid A in formalin-fixed, paraffin-embedded tissues and to expand the spectrum of the amyloid proteins analyzed by this method, we studied formalin-fixed, paraffin-embedded amyloid-containing tissues obtained from patients with the following types of amyloidosis: amyloid A, light chain, transthyretin, calcitonin, beta2 microglobulin, and senile seminal vesicle. The tissue samples were deparaffinized, processed, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot technique. Only specimens from patients with amyloid A amyloidosis gave protein bands: a single 8.5-kd band in lanes of all tissues studied, except thyroid tissue, which displayed two bands of about 5 and 10 kd. Other types of amyloid failed to show any protein band. These findings suggest that the Shtrasburg method is sensitive, specific, and reliable and may have an important role in the diagnosis of amyloidosis.

Hemodialysis-related arthropathy. A prospective MR study with SE and GRE sequences.

A prospective examination of hemodialysis-related arthropathy by MR imaging with SE and GRE sequences was undertaken to verify whether the low signal intensity on T2-weighted SE images is caused by a paramagnetic susceptibility effect derived from amyloid or from hemosiderin. Twenty-seven joints (19 hip joints, 6 discovertebral joints of 5 lumbosacral spines and 1 cervical spine, 2 shoulder joints) in 14 patients undergoing long-term hemodialysis (duration 10-22 years, mean 17) were examined with SE T1-weighted, T2-weighted and GRE T2*-weighted sequences. The signal intensity of the intraarticular and periarticular masses of the involved joints was basically isointense to the muscle in all 3 pulse sequences. No significantly low signal area was found on GRE T2*-weighted images. The low signal areas in the hemodialysis-related arthropathy on SE T2-weighted images are not caused by a paramagnetic susceptibility effect, but probably by the hypocellular and fibrous nature of amyloid-containing tissues. Therefore the GRE sequence can be helpful for differentiating hemodialysis-related amyloid arthropathy from chronic hemarthrosis or juxta-articular brown tumor.

Determination of amyloid type by ELISA using milligram amounts of tissue

A new procedure was developed for isolation and immunochemical identification of amyloid proteins. This procedure involves extraction of amyloid proteins from tissues with aqueous acidic acetonitrile, their purification by HPLC and identification by ELISA. In this way the type of amyloid proteins was defined in amyloid-containing tissues obtained from 15 patients. The technique is simple, rapid and enables typing of amyloid proteins using only milligram amounts of tissue. This is in contrast to the conventional amyloid isolation and chemical identification technique which is laborious, time consuming and requires gram amounts of tissues. The procedure may enable the identification of the type of amyloid in biopsy specimens and thus help in evaluation of prognosis and determination of the therapeutic policy.

Fibrils from Synthetic Amyloid-Related Peptides Enhance Development of Experimental AA-Amyloidosis in Mice

Amyloid enhancing factor is an incompletely characterized activity of extracts from many amyloid-containing tissues and which greatly shortens the preamyloidotic phase during experimental induction of AA-amyloidosis. In this communication we show that amyloid-like fibrils made in vitro from synthetic peptides, corresponding to segments of amyloid fibril proteins, have amyloid enhancing factor-like activity. Thus, there is a possibility that amyloid enhancing factor activity depends on small fibrils serving as nucleation centers for fibril elongation.

Isolation and Characterization of Amyloid Proteins Using Milligram Amounts of Amyloid - Containing Tissue

Abstract A new procedure is developed for extraction and purification of amyloid proteins from milligram amounts of amyloid-containing tissue. The procedure involves extraction of amyloid proteins with acidic agueous acetonitrile and their purification by size-exclusion high-performance liquid chromatography (HPLC). The molecular weight and type of isolated amyloid proteins were determined by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting technique. The developed procedure is more rapid and requires significantly smaller amount of tissue material (about 10 mg), comparing with the conventional amyloid isolation technique performed by using gram amounts of autopsy specimen. The techniques applied presently may be useful for precise determination of amyloid type in biopsies.

Unmasking antigen determinants in amyloid.

Rehydrated paraffin sections of formalin-fixed, amyloid-containing tissues were treated with denaturing agents (guanidine and urea) and reducing agents (DDT and mercaptoethanol) before immunostaining, in an attempt to expose antigenic determinants hidden in the rigid structure of amyloid fibrils. Pre-treatment overnight with 6 M guanidine or urea was found beneficial, especially in specimens from familial amyloid polyneuropathy of the Portuguese type. Addition of reducing agents had no major effect. Modifications of this method may be useful in unmasking other antigens that are polymerized or considered destroyed by fixation and paraffin embedding.

Amyloid-enhancing factor (AEF) in the pathogenesis of AA-amyloidosis in the hamster.

AA-amyloidosis was induced in hamsters receiving amyloid-enhancing factor (AEF) by daily subcutaneous injection with either an aged casein solution or casein supplemented with lipopolysaccharide (LPS). Both amyloid inducers gave similar results with respect to amyloid development in spleen, liver and kidneys and to serum amyloid A (SAA) concentrations and plasma cathepsin D activities. AEF was isolated from amyloid-containing tissue by the method described by Hol et al. (1985), and amyloid-enhancing material was also extracted from isolated hamster amyloid fibrils by intensive sonification. This fibril-derived amyloid-enhancing material lacked typical green birefringence after staining with Congo red and appeared as amorphous material on electron microscopy. AEF shortened the pre-amyloid phase for splenic and hepatic amyloid development and also the subsequent interval before renal amyloid deposition. This indicates that endogenous AEF, unlike passively transferred preformed AEF, is not distributed throughout the body and is probably generated at the site of amyloid deposition. Moreover, these results suggest that amyloid deposition in the kidneys, like that in the spleen and liver, involves an AEF-dependent pathway. Thus redistribution of amyloid is probably not an important cause of renal amyloid involvement. In addition to the reduction in the lag phase for splenic and hepatic amyloid deposition, AEF also speeds the changes in SAA concentration and plasma cathepsin D activity. This indicates that AEF accelerates rather than eliminates the pre-amyloid phase.

Antigens Characteristic for Amyloid of Man and Rabbit

Summary Rabbit antisera to human amyloid tissue gave a positive indirect immunofluorescence test with sections of amyloid-containing organs. Absorption experiments indicated that antibodies to previously described amyloid-characteristic components participated in this test. Evidence was presented that the amyloid antigen described by Cathcart et al. is probably identical with the previously described lipoprotein component of amyloid-containing tissues. By means of guinea pig immune sera, a lipoprotein antigen was identified in amyloid-containing rabbit organs. An identical antigen was detected in normal rabbit serum but not in normal rabbit organs.

The cause of the green polarization color of amyloid stained with Congo red.

Experiments done with Congo red crystals and with Congo red deposits polished in a single direction by a glass wheel have shown that the appearance of green polarization color primarily depends on near-perfect parallel alignment of the dye particles. The green polarization color was seen only in the deposits which showed a clear transition from red to colorless when examined for dichroism. Another factor was found to be the thickness of the object, as the green polarization color was not present in too thick or too thin sections of amyloid-containing tissues stained with Congo red.