Reinhold P. Linke

Expression and function of serum amyloid A, a major acute-phase protein, in normal and disease states.

Serum amyloid A (SAA), the precursor protein in inflammation-associated reactive amyloidosis (AA-type), is an acute phase reactant whose level in the blood increases in response to various insults. It is expressed in the liver, but its physiological role is not well understood. Recently, a broader view of SAA expression and function has been emerging. Expression studies show local production of SAA proteins in histologically normal, atherosclerotic, Alzheimer, inflammatory, and tumor tissues. Binding sites in the SAA protein for high density lipoproteins, calcium, laminin, and heparin/heparan-sulfate were described. Adhesion motifs were identified and new functions, affecting cell adhesion, migration, proliferation and aggregation have been described. These findings emphasize the importance of SAA in various physiological and pathological processes, including inflammation, atherosclerosis, thrombosis, AA-amyloidosis, rheumatoid arthritis, and neoplasia. In addition, recent experiments suggest that SAA may play a “housekeeping” role in normal human tissues.

Highly sensitive diagnosis of amyloid and various amyloid syndromes using Congo red fluorescence

Abstract In order to find how best to diagnose amyloid deposits as early as possible, the sensitivity of three different methods that can be applied to the diagnosis of amyloid in tissue sections have been compared: the Congo red staining method (CR), the combination of CR and immunocytochemistry (CRIC) and Congo red fluorescence (CRF). Tissue blocks were available from 25 patients, including 11 with immunohistochemically distinct and 3 with chemically undefined amyloid diseases. The results revealed (a) that CRF is more sensitive than either CR or CRIC, as shown qualitatively and quantitatively, (b) that CRF can therefore be utilized to track down even minute amyloid deposits, which can be missed by the other two methods; (c) that the specificity of CRF and CRIC is secured on double-stained sections by the demonstration of green birefringence (GB) of the CRF-marked and IC-marked areas; (d) that CRF can be performed on the spot by just changing the light source; and (e) that CRF is not hampered by the congruent IC chromogen overlay, which ensures the specific classification of the amyloid deposits as applied to different amyloid classes. In conclusion, CRF was demonstrated to be the most sensitive method for direct diagnosis of amyloid in tissue sections. This method can, therefore, allow the earliest diagnosis and classification of amyloid, which is a good basis for an amyloid class-specific therapy while organ damage is still minimal.

The classification of amyloid deposits in clinicopathological practice

A series of 104 biopsy cases with histopathological proof of amyloid, submitted to our department of pathology over the last 19 years, were re‐examined. The survey investigated the medical indication for surgery, the origin and quality of the biopsy and the clinical information as documented on the request form for histopathological examination and in hospital records. Amyloid deposits were classified using antisera directed against five major amyloid fibril proteins, i.e. AA, ATTR, Aλ, Aκ and Aβ2M and optimal conditions were sought for the reliable and early characterization of amyloid disease in clinicopathological practice. This survey revealed that 98% of the biopsy cases already suffered from a disease which was either a cause or a result of amyloidosis. In only 2% of the biopsy cases was amyloidosis detected without any clinical indication. Immunohistochemical classification of the amyloid deposits and comparison with hospital records demonstrated diagnostic pitfalls such as immunostaining of amyloid by two or more antibodies recognizing different fibril proteins, and disagreement between immunohistochemical typing of amyloid and the initial clinical diagnosis. Based on these observations we assume that the characterization of amyloid disease and its biological significance is impossible in clinicopathological practice without clinical information or without immunohistochemical classification of the fibril protein in biopsy specimens. Different aspects of histopathological detection of AA‐ and AL‐amyloidosis are discussed.

Selection of D-Amino-Acid peptides that bind to Alzheimer's disease amyloid peptide A beta(1-42) by mirror image phage display

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimers disease amyloid peptide Aβ(1–42). A randomized 12‐mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Aβ(1–42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D‐pep) was shown to bind Aβ(1–42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimers disease, amyloid plaques and leptomeningeal vessels containing Aβ amyloid were stained specifically with a fluorescence‐labeled derivative of D‐pep. Fibrillar deposits derived from other amyloidosis were not labeled by D‐pep. Possible applications of this novel and highly specific Aβ ligand in diagnosis and therapy of Alzheimers disease are discussed.

Inhibition of the oxidative burst response of N-formyl peptide-stimulated neutrophils by serum amyloid-A protein

Strong binding of the acute phase protein serum amyloid-A (SAA) to human neutrophils was found using flow cytometry. This binding was shown to be functionally relevant with respect to the oxidative burst reaction assayed on N-formyl peptide-stimulated neutrophils by the intracellular oxidation of non-fluorescent dihydrorhodamine to fluorescent rhodamine 123. The results show reduction of the oxidative burst response by isolated SAA (and recombinant SAA2). Inhibition was also demonstrated by acute phase as compared to normal human serum. This inhibitory effect was abolished by the purified monoclonal anti-amyloid A antibody mc29, strongly suggesting that SAA counteracts neutrophil responses to cytokines or bacterial products. This newly recognized function of SAA may help to prevent oxidative tissue destruction.

Diagnosis and immunohistochemical classification of systemic amyloidoses : Report of 43 cases in an unselected autopsy series

Abstract Fourty-three cases of systemic amyloidosis were identified in an unselected autopsy series from our institute (6305 autopsies between 1979 and 1993) and classified immunohistochemically by means of a panel of antisera directed against five major amyloid fibril proteins. Amyloid A (AA) amyloidosis was the most common type, being found in 21 cases (48.8%). Transthyretin-derived (ATTR) amyloidosis was present in 11 cases (25.6%), and immunoglobulin light chain-derived (AL) amyloidosis in 10 cases (23.3%). A single case (2.3%) contained deposits of more than one type of systemic amyloid. AA amyoloidosis was associated with chronic inflammatory or infectious diseases (81%), malignant tumours (19%) or both (9.5%). Immunoglobulin light chain-derived amyloidoses were associated with myeloma (50%) or primary (idiopathic; 50%). In AA and AL amyloidosis the kidney was the organ most frequently involved. ATTR amyloid affecting mostly the heart and lungs presented as senile systemic amyloidosis. Systemic amyloidosis was the cause of death in 5 cases (12%) and caused symptoms in 17 cases (39%). Our results suggest that most cases can be classified by using a panel of sensitive and specific antibodies against five major amyloid fibril proteins. This technique may make amyloid type-specific therapy possible for AL amyloid patients who do not have evidence of an underlying plasma cell dyscrasia.

High-dose melphalan with autologous stem cell transplantation after VAD induction chemotherapy for treatment of amyloid light chain amyloidosis: a single centre prospective phase II study

Amyloid light chain (AL) amyloidosis is the result of a clonal plasma cell expansion, in which monoclonal light chains transform to amyloid deposit in various tissues and can lead to organ dysfunction and organ failure. The median survival of patients with AL amyloidosis without therapy is 10–14 months. With high‐dose melphalan (HDM) and autologous stem cell transplantation (ASCT), haematological and clinical remission rates of up to 50% of treated patients have been reported from phase II studies. HDM followed by ASCT appears to prolong survival in patients, if haematological remission can be reached. In this phase II study, we evaluated vincristine, adriamycin and dexamethasone (VAD) as induction chemotherapy prior to stem cell mobilization and HDM with ASCT. The regimen was, in general, feasible in patients with AL amyloidosis, but VAD chemotherapy had a considerable World Health Organization (WHO) grade III–IV toxicity (25%) and mortality (7%) rate. VAD pretreatment did not interfere with stem cell mobilization and HDM with ASCT was possible in 86% of patients. The overall treatment efficacy was comparable with reported results of HDM and ASCT without preceding chemotherapy. We could not show an additional benefit of VAD induction in terms of increasing haematological response rate; however the 13% mortality rate after HDM and ASCT in our series was lower than the previous report.

Expression of serum amyloid A, in normal, dysplastic, and neoplastic human colonic mucosa: implication for a role in colonic tumorigenesis.

Serum amyloid A (SAA) is an acute phase reactant, whose level in the blood is elevated in response to trauma, infection, inflammation, and neoplasia. Elevated levels of SAA in the serum of cancer patients were suggested to be of liver origin rather than a tumor cell product. The role of SAA in human malignancies has not been elucidated. We investigated the expression of SAA at various stages of human colon carcinoma progression. Nonradioactive in situ hybridization applied on paraffin tissue sections from 26 colon cancer patients revealed barely detected SAA mRNA expression in normal looking colonic epithelium. Expression was increased gradually as epithelial cells progressed through dysplasia to neoplasia. Deeply invading colon carcinoma cells showed the highest levels of SAA. Expression was also found in colon carcinoma metastases. Cells of lymphoid follicles of the intestinal wall, inflammatory cells, ganglion cells, and endothelial cells, also expressed SAA mRNA. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. RT-PCR analysis confirmed the expression of the SAA1 and SAA4 genes in colon carcinomas, expression that was barely detectable in normal colon tissues. These findings indicate local and differential expression of SAA in human colon cancer tissues and suggest its role in colonic tumorigenesis.

Classification of Amyloid Syndromes from Tissue Sections Using Antibodies Against Various Amyloid Fibril Proteins: Report of 142 Cases

To identify the chemical nature of amyloid fibril proteins in tissue sections, various formalin-fixed organs from 142 patients with amyloid were investigated with a panel of antisera directed against different purified amyloid fibril proteins from representative generalized amyloid syndromes.

Immunohistology of islet amyloid polypeptide in diabetes mellitus: Semi-qantitative studies in a post-mortem series

Immunoreactivity for islet amyloid polypeptide (IAPP) in the islets of Langerhans of non-insulin-dependent diabetic patients and non-diabetic patients of a non-selected post-mortem series was studied with a new polyclonal IAPP antibody. Out of 133 patients examined, 124 exhibited immunoreactivity for IAPP. Immunoreactivity was localized intra- and extracellularly and was limited to the islets of Langerhans. No extracellular immunoreactivity was observed in amyloid-negative cases. Co-localization of insulin and IAPP in the same islet-cells was verified by double staining with monoclonal insulin and polyclonal IAPP antibodies. Of 100 patients with non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid, 98 exhibited IAPP-positive deposits and 71 exhibited intracellular immunoreactivity. Evaluation of intracellular immunoreactivity and degree of islet amyloid deposition in cases of overt NIDDM revealed an inverse relationship, in that intracellular IAPP immunoreactivity were reduced in patients with developing islet amyloid deposition. Our data are consistent with the hypothesis of primaryβ-cell dysfunction leading to amyloid formation, with subsequent disturbance ofβ-cell homeostasis.