Atypical familial motor neuropathy in patients with mutant TTR Ile68Leu.

Two sisters from an Italian family shared progressive motor symptoms, preceding the onset of sensory and autonomic disturbances. The familial occurrence of axonal and slowly progressive polyneuropathy led us to consider these patients as candidates for TTR molecular analysis. We found a missense mutation causing Ile68Leu TTR substitution in both. The aims of this work are to report the possibility of a motor onset of amyloid polyneuropathy and to suggest the search for TTR mutations in familial cases of axonal polyneuropathy. Second, to stress the possible occurrence of amyloid within the spinal canal as the potential pathogenesis and responsible for motor presentation.

Histopathologic and immunophenotypic characterization of extramedullary plasmacytomas in nine cats.

To contribute to the existing body of knowledge in the literature on the apparently rare extramedullary plasmacytoma in cats, lymphoid tumors with plasmacytic cellular morphology taken from nine cats were examined. The paraffin-embedded material was investigated by standard hematoxylin and eosin, and special staining techniques (Giemsa, Congo-red, and periodic acid-Schiff reaction). The tumors also were examined immunohistochemically for the presence of immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin light chains (lambda, kappa), various amyloid proteins, and FeLV-antigen (p27 protein). An immunoglobulin-producing tumor of plasmacellular origin (extramedullary plasmacytoma [EMP]) could be diagnosed in all cases on the basis of immunohistochemical light-chain expression. All but one of the neoplasms occurred in the skin of older, predominantly male cats. As in humans and dogs, the following types could be identified according to their morphologic features: mature type (two), cleaved type (two), asynchronous type (four), and polymorphous type (one). The tumor tissue of three cats revealed amyloid deposits, which were immunohistochemically diagnosed as ALlambda-amyloid in all three cases.

[Hereditary amyloidoses associated with transthyretin mutations]. / Transthyretinassoziierte hereditäre Amyloidosen.

Hereditary amyloidoses form a clinically and genetically heterogeneous group of autosomal-dominantly inherited diseases characterized by the ubiquitous extracellular deposit of fibrillary aggregated proteins. Main components of these unsolvable deposits are physiologic proteins that became amyloidogenic through genetically determined conformation changes resulting in an increase in beta-sheet structures. In the vast majority of cases, the offending protein is variant transthyretin (TTR), of which over 80 mutations are known. Among these, substitution of valine by methionine in position 30 (TTR-Met30) is the most commonly encountered. In typical cases, TTR amyloidoses present with polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, and gastrointestinal features, occasionally accompanied by vitreous opacities and renal insufficiency. Rarely, involvement of the leptomeningeal or meningovascular structures dominates the clinical picture. The clinical expression is highly variable, with many atypical manifestations. Asymptomatic mutations have recently been identified. The age of onset varies greatly between early adulthood and old age. Late-onset atypical manifestations and occurrence of asymptomatic carriers render identification of affected family members difficult despite autosomal-dominant inheritance. Orthotopic liver transplantation (OLT) is the only effective therapy available today. This OLT stops progression of the disease, which is otherwise invariably fatal, by removal of the main production site of the amyloidogenic protein. However, cardiac involvement may progress after OLT for unknown reasons. The indication for OLT and its success depend on the grade of cardiovascular and autonomic dysfunction at the time of surgery, age, comorbidity, and type of mutation. Alternative treatment modalities with drugs stabilizing the native tetrameric conformation of TTR, inhibiting fibril formation or breaking beta-sheet structures, are currently being intensively studied.

Cutaneous lymphatic amyloid deposits in "Hungarian-type" familial transthyretin amyloidosis: a case report.

Multiple transthyretin (TTR) mutations have recently been identified and implicated in the development of familial systemic amyloidoses, but early diagnosis of these disorders is still largely unresolved. We investigated the presence and tissue distribution of TTR-derived amyloid in skin biopsies of a 59-year-old woman carrying the "Hungarian-type" mutation of TTR (Asp18Gly). Clinical symptoms involved severe central nervous system dysfunction without signs of polyneuropathy, also referred to as the "central form" of TTR-related systemic amyloidosis. Skin biopsy was also evaluated as a tool in order to diagnose this type of TTR amyloidosis. Biopsy samples were collected from the infra-axillary region. Light microscopy using Congo red and polarized light was used to diagnose amyloid deposits. Subsequently, electron microscopic analysis was performed to correlate the amyloid deposits with vicinal dermal structures. The amyloid class was determined by means of immunocytochemistry. TTR amyloid was primarily localized to lymphatic microvessels in the present case, whereas arterioles were devoid of TTR amyloid deposits. In addition, the well-known association of TTR amyloid with neural structures along the erector pilorum and around the sebaceous and serosal (sweat) glands was also evident. Electron microscopic analysis of amyloid deposits revealed characteristic amyloid fibrils that were irregular in shape, and exhibited a heterogeneous density and a random deposition pattern. Immunocytochemistry confirmed the cutaneous accumulation of TTR amyloid. In conclusion, amyloid deposits were abundantly present in the skin of a patient with "Hungarian-type" TTR amyloidosis; skin biopsy seems to be appropriate for the diagnosis of this disorder. We showed that besides the erector pilorum, sweat glands and nerve terminals, lymphatic microvessels are also severely infiltrated by TTR amyloid. Whether these pathological alterations can exclusively be found in "Hungarian-type" TTR amyloidosis should still be investigated. If such changes are not specific for the Asp18Gly mutation, they may be considered as diagnostic markers for "central" TTR amyloid disorders.

Transthyretin-associated neuropathic amyloidosis. Pathogenesis and treatment.

Hereditary amyloidoses form a clinically and genetically heterogeneous group of autosomal dominantly inherited diseases characterized by the deposit of insoluble protein fibrils in the extracellular matrix. They typically present with polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, and cardiomyopathy and gastrointestinal features, occasionally accompanied by vitreous opacities and renal insufficiency. Other phenotypes are characterized by nephropathy, gastric ulcers, cranial nerve dysfunction, and corneal lattice dystrophy. Rarely, involvement of the leptomeningeal or cerebral structures dominates the clinical picture. The age at onset is as early as 17 and as late as 78 years. The basic constituents of amyloid fibers are physiologic proteins that have become amyloidogenic through genetically determined conformation changes. Mutated transthyretin (TTR), formerly termed prealbumin, is the most frequent offender in hereditary amyloidosis. Orthotopic liver transplantation (OLT) stops the progression of the disease, which is otherwise invariably fatal, by removing the main production site of amyloidogenic protein. The indications for OLT and its success depend on the grade of cardiovascular and autonomic dysfunction at the time of surgery, age, comorbidity, and type of mutation. Alternative treatment modalities with drugs stabilizing the native tetrameric conformation of TTR and inhibiting fibril formation are currently being studied.

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response.

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP. In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.

Amyloid in a multiple sclerosis lesion in clearly of Alambda type.

In rare multiple sclerosis cases amyloid is deposited in demyelinated plaques. In one such case amyloid was examined immunohistochemically with a panel of antibodies directed against different amyloid types. The amyloid was classified as the Alambda type produced by a local monoclonal B cell population.

Recombinant versus natural human 111In-beta2-microglobulin for scintigraphic detection of Abeta2m amyloid in dialysis patients.

BACKGROUND: We previously introduced scintigraphy with 131I-labeled beta2-microglobulin (beta2m), purified from uremic hemofiltrate, that is, "natural" beta2m, to specifically detect beta2m-associated amyloidosis (Abeta2m) in hemodialysis (HD) patients. METHODS: To improve the safety and resolution of the scan, we covalently bound the chelator diethylenetriaminepentaacetic acid to natural beta2m to allow radiolabeling with 111In. In a second step, we generated and evaluated the usage of recombinant human beta2m (rhbeta2m) for scintigraphy. RESULTS: Using natural 111In-labeled beta2m, eight patients on HD for 0 to 17 years, without evidence of Abeta2m, were scanned. Whole-body scintigraphy at 48 to 72 hours postinjection revealed no significant tracer accumulation over joint regions. In contrast, nine patients on HD for 10 to 21 years with clinical, radiological, or histologic (N = 4) evidence of Abeta2m showed selective tracer uptake over various joint regions. Tracer accumulation in visceral organs, which could not be related to tracer elimination or metabolism, was not detected. Compared with the previous 131I beta2m scan, scintigraphy with 111In-labeled beta2m offered highly improved image contrast, increased sensitivity, and a 50 to 70% reduction of the radiation exposure. Scanning with 111In-labeled recombinant human beta2m was performed in six patients: No significant tracer accumulation was observed over joint regions in two patients on short-term HD without evidence of Abeta2m; in contrast, local tracer accumulations similar to those observed with natural beta2m could be demonstrated in four long-term (10 to 27 years) HD patients with clinical, radiological, and histologic (N = 1) evidence of Abeta2m. CONCLUSION: Scintigraphy for Abeta2m with 111In-labeled rhbeta2m provides a homogenous and safe recombinant protein source and leads to enhanced sensitivity and lower radiation exposure.

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

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.

Production of recombinant human beta2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis.

Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.

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.

Cerebrovascular involvement in systemic AA and AL amyloidosis: a clear haematogenic pattern.

Amyloid deposits in cerebral vessels are common in beta-amyloid diseases (Alzheimer's disease, congophilic amyloid angiopathy, Down's syndrome and hereditary cerebral amyloidosis with haemorrhage of the Dutch type). We report of 20 autopsies on patients who had died with systemic amyloidosis of the AA, Alambda and Akappa types: the brains were examined for the occurrence of amyloid. Vascular amyloid was detected in choroid plexus (in 17 of 20 cases), infundibulum (5 of 8), area postrema (6 of 11), pineal body (3 of 7) and subfornical organ (2 of 3), but not in cortical and leptomeningeal vessels. Immunohistochemical classification of the cerebral amyloid and the systemic amyloid syndrome showed identity proving the same origin of both. The distribution is indicative of a haematogenic pattern of amyloid deposition in systemic amyloidosis and is different from that in Alzheimer's, prion, ATTR and cystatin C diseases. It corresponds to areas of the brain with a "leaky" blood-brain barrier. Additionally, all the cases with AA amyloidosis exhibited an Abeta coreactivity in choroid plexus vessels. In one exceptional case, Abeta reactivity of AA amyloid also occurred outside of the brain.

Clinical benefits of diagnosing incipient AA amyloidosis in pediatric rheumatic diseases as estimated from a retrospective study.

OBJECTIVE: The diagnosis of AA amyloidosis could not be made in eight patients with pediatric rheumatic diseases as later verified employing the more sensitive combination of Congo red and additional immunocytochemistry (CRIC). The objective of this paper is to estimate the benefit of CRIC by reevaluating the historical charts with respect to the question as to which of the diagnostic and therapeutic measures would have been altered if the correct diagnosis had been known at the time of the primary biopsy. METHODS: All subsequent biopsies of eight children with historically missed AA amyloidosis in their primary biopsies were retrieved, together with the historical data including the Congo red stains of the biopsies. The biopsies were reexamined blindly for the presence of amyloid and the results were compared with the historical data concerning diagnostic and therapeutic measures. RESULTS: Using CRIC, AA amyloidosis could be identified an average of approximately three years earlier as compared to the historical data. This gain in time would certainly have altered the diagnostic as well as the therapeutic options, i.e. 10 out of 21 biopsies would have been spared and the earlier diagnosis would have initiated more significant antiinflammatory therapy. CONCLUSION: Very early detection of amyloid reduces the diagnostic burden and unveils an option for a consequent antiinflammatory therapy very early in the course of AA amyloidosis.