Correlations between the morphological and clinical findings in a patient recovering from secondary generalised amyloidosis with renal involvement

Summary We report light- and electron microscopic findings in glomerular amyloidosis (secondary amyloidosis), which occured after recurrent empyema of the pleura. After healing of the empyema, the clinical symptoms disappeared, over a period of eight years.During the acute stage of the disease (grade II–III amyloidosis) when the nephrotic syndrome was present, amyloid deposits were seen in the mesangium and on both sides of the basement membrane of the glomerular capillaries. Furthermore, denuded basement membrane areas showing the passage of amyloid into the urinary space, and invaginations of the podocyte by straightened amyloid fibrils were found. After clinical recovery (except for a trace of proteinuria), the renal amyloidosis had electronmicroscopically transformed from an active into an inactive or resting form, while the amount of amyloid present was almost the same. In the areas of amyloid deposits, reparative changes were observed, especially in the area of the mesangial cells and of the podocytes. The podocytes were separated from the persisting amyloid deposits by newly formed basement membrane material.Supported by the Deutsche Forschungsgemeinschaft     

Clinical and histological characteristics of renal AA amyloidosis: a retrospective study of 68 cases with a special interest to amyloid-associated inflammatory response

We retrospectively reviewed the clinicopathological features of a series of 68 renal AA amyloidosis observations collected between 1990 and 2005. The amyloidogenic disease was a chronic infection (40.8%), a chronic inflammation (38%), a tumor (9.9%), a hereditary disease (9.9%), or was undetermined in 1.4% of cases. Nephrotic syndrome and renal insufficiency were noted in 63.1% and 75% of patients, respectively. The distribution pattern of glomerular amyloid deposits was mesangial segmental (14.7%), mesangial nodular (26.5%), mesangiocapillary (32.3%), and hilar (26.5%). Glomerular form was observed in 80.9% of cases and vascular form in 19.1%. AA amyloidosis-related inflammation was noted in 30 patients (44.1%) and appeared as a multinucleated giant cell reaction (27.9%) or a glomerular inflammatory infiltrate (25%), including glomerular crescents (17.6%). At the end of follow-up, 26 patients (38.2%) showed end-stage renal disease. The clinical presentation of glomerular and vascular forms was distinct with a clear predominance of proteinuria in glomerular form. Inflammatory reaction was preferentially observed in biopsies with a codeposition of immunoglobulin chains and/or complement factors in AA amyloid deposits. The distribution pattern of glomerular amyloid deposits and glomerular inflammatory reaction were independent factors influencing proteinuria level. Tubular atrophy, abundance, and distribution pattern of glomerular amyloid deposits at the time of biopsy were independent predictors of renal outcome. In conclusion, the glomerular involvement appeared as the determining histological factor for clinical manifestations and outcome of renal AA amyloidosis. AA amyloidosis-related inflammation could partly result from an immune response directed against AA fibrils and could induce amyloid resolution and crescents.     

Glomerular crescents in renal amyloidosis: An epiphenomenon or distinct pathology?

There have been several reports of cases of renal amyloidosis with glomerular crescents. However, it is not clear whether the association is fortuitous or pathogenic related. The present study analyzed 105 cases of renal amyloidosis (61 autopsy cases and 44 biopsy cases) and found glomerular crescents in 14 (13.3%) cases. Among the 14 cases with crescents, a female predominance was noted (male: female, 3: 11) and rheumatoid arthritis was the most common primary disease of amyloidosis. Immunohistochemical analysis demonstrated amyloid protein of AA type in 12 cases. According to the histologic classification, there were 11 cases of mesangial nodular type, which was almost exclusively accompanied by AA amyloid deposition. Of note, the incidence of crescents neither correlated with the extent of amyloid deposition nor the presence of nephrotic syndrome. By contrast, localization of amyloid deposition was closely related to crescent formation. Moreover, electron microscopic observation displayed rupture of the glomerular basement membrane at the site of amyloid deposition. Our results indicated that glomerular crescents were more frequently associated with renal amyloidosis than previously appreciated. Rupture of the fragile glomerular basement membrane by amyloid deposition, as revealed by immunostaining and electron microscopy, may be the mechanism of crescent formation. We suggest that glomerular crescents are a distinct pathology associated with renal amyloidosis, not fortuitous conditions.     

Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis

The human serum protein transthyretin (TTR) is highly fibrillogenic in vitro and is the fibril precursor in both autosomal dominant (familial amyloidotic polyneuropathy [FAP] and familial amyloidotic cardiomyopathy [FAC]) and sporadic (senile systemic amyloidosis [SSA]) forms of human cardiac amyloidosis. We have produced mouse strains transgenic for either wild-type or mutant (TTRLeu55Pro) human TTR genes. Eighty-four percent of C57BI/6xDBA/2 mice older than 18 months, transgenic for the wild-type human TTR gene, develop TTR deposits that occur primarily in heart and kidney. In most of the animals, the deposits are nonfibrillar and non-Congophilic, but 20% of animals older than 18 months that bear the transgene have human TTR cardiac amyloid deposits identical to the lesions seen in SSA. Amino terminal amino acid sequence analysis and mass spectrometry of the major component extracted from amyloid and nonamyloid deposits revealed that both were intact human TTR monomers with no evidence of proteolysis or codeposition of murine TTR. This is the first instance in which the proteins from amyloid and nonfibrillar deposits in the same or syngeneic animals have been shown to be identical by sequence analysis. It is also the first time in any form of amyloidosis that nonfibrillar deposits have been shown to systematically occur temporally before the appearance of fibrils derived from the same precursor in the same tissues. These findings suggest, but do not prove, that the nonamyloid deposits represent a precursor of the fibril. The differences in the ultrastructure and binding properties of the deposits, despite the identical sizes and amino terminal amino acid sequences of the TTR and the dissociation of deposition and fibril formation, provide evidence that in vivo factors, perhaps associated with aging, impact on both systemic precursor deposition and amyloid fibril formation.     

Immunogold quantitation of immunoglobulin light chains in renal amyloidosis and kappa light chain nephropathy.

By quantitative immunoelectron microscopy using protein A-gold, the authors compared the content and distribution of immunoglobulin light chain (LC) antigens in glomeruli from 11 cases of renal amyloidosis with that in two cases of kappa LC glomerulopathy and two cases of diabetic glomerulosclerosis. In a supplementary study and using a similar immunogold technique, the authors identified amyloid A in deparaffinized renal tissue from three of the 11 cases of renal amyloidosis. Each patient had similar clinical manifestations (chronic renal failure with proteinuria) and similar glomerular morphology (thickened glomerular basement membranes and nodular expansion of the mesangium). In 12 cases (10 amyloid, 2 kappa LC), immunoelectron microscopy localized LC antigens over the glomerular deposits and allowed indirect tissue quantitation of each LC antigen to the various cellular and interstitial compartments. In 6 of the 11 cases of renal amyloidosis, the amyloid labeled only for lambda, and in one, only for kappa. In one patient with Waldenström''s macroglobulinemia, who had a biclonal gammopathy, both LC were identified in the amyloid. In two cases, both of whom had a history of chronic suppurative lung disease, both LC antigens as well as amyloid A were localized to the amyloid fibrils. In only one case, in which glomerular amyloid labeled for amyloid A, the amyloid did not label for either LC. Whereas lambda LC-derived fibrils often appeared as spicules in the glomerular subepithelial space, other amyloid deposits usually accumulated in the subendothelial zone and did not form spicules. The epimembranous location of spicules suggested that the amyloid precursor protein transformed into amyloid fibrils after filtration into the urinary space. Presence of epimembranous spicules may explain the more severe proteinuric renal failure and the more rapid progression to glomerulosclerosis described in primary amyloidosis.     

THE FINE STRUCTURE OF GLOMERULAR EPITHELIAL CELLS IN EXPERIMENTAL RENAL AMYLOIDOSIS

The fine structure of the three types of glomerular cells, especially glomerular epithelial cells was studied In experimental renal amyloidosis induced by both prolonged sensitization of focus antigens and casein.
The presence of cytoplasmic invaginations which contained a bundle of amyloid fibrils in almost parallel array and intracytoplasmlc membrane-surrounded amyloid fibrils were observed in the glomerular epithelial cells. They are quite comparable to those which were demonstrated in reticuloendothelial cells In other reports. The intracytoplasmlc invaginations opened at the plasma membrane either in the urinary space or in the basement membrane. By processing serial sections on the minimal deposition of amyloid, some of the Intracytoplasmlc membrane-surrounded amyloid fibrils were found to be connected with parallel arrayed extracellular amyloid fibrils.
Amyloid accumulated usually In large amount In mesangial matrix and along the basement membrane adjacent to the mesangium, but isolated depositions of amyloid, minimal though definite, were sometimes observed in the subepithelial and subendothelial areas, and in the epithelial cells far from the mesangium.     

Morphologic and clinical correlates in renal amyloidosis

Morphologic studies and clinical correlations were undertaken in 59 patients with renal amyloidosis. Spicularly arranged amyloid deposits in the glomerular capillary wall were found in all clinical groups but were more frequent and more extensive in primary amyloidosis and multiple myeloma. The severity of proteinuria correlated with the presence of spicules and podocyte destruction rather than with the amount of amyloid in the glomerulus. The spicules were associated with morphologic and clinical evidence of rapid amyloid deposition and a fulminant clinical course. The absence of spicules and the presence of extensive new basement membrane material may produce basement membrane thickening, lamination, and double capillary wall contours, which are associated with mild proteinuria and, rarely, resolution of amyloidosis. Nodular or mixed nodular-diffuse patterns of glomerular amyloid deposits were more frequent in patients with secondary amyloidosis and a longer clinical course. Renal failure generally corresponded to severe glomerular amyloidosis and tubular atrophy. However, a relatively precipitous, usually irreversible decrease in renal function frequently occurred in patients with renal amyloidosis and did not always have a morphologic explanation. The duration of life from the time of biopsy to death in patients with primary amyloidosis (nine months) was markedly shorter than in those with secondary amyloidosis (more than 50 months).     

Tissue distribution of amyloid deposits in Abyssinian cats with familial amyloidosis

The tissue distribution of amyloid deposits was studied in 15 related Abyssinian cats with familial amyloidosis. There was interstitial medullary amyloidosis in the kidneys of all 15 cats but only 11 had detectable glomerular involvement. The thyroid glands, stomach and colon were affected in all cats examined. Most of the cats also had amyloid deposits in the small intestine, spleen, heart, adrenals, pancreas, liver, lymph nodes and bladder. In 50 per cent or fewer of the cats examined, there was involvement of the parathyroids, lung and gonads. The central nervous system was not involved in any of the 3 cats evaluated. In 8 of the cats, no concurrent inflammatory disease could be detected. The tissue distribution of amyloid deposits resembled that found in other breeds of domestic cats with systemic amyloidosis. Despite the wide tissue distribution of amyloid deposits, clinical signs were related to renal amyloidosis. Familial amyloidosis in the Abyssinian cat may represent a valuable spontaneous animal model for the study of Familial Mediterranean Fever in man and the pathogenesis of reactive amyloidosis in general.     

Ultrastructural studies on the evolution of amyloidosis in the cyclic hematopoietic (CH) dog

Summary Electron microscopy studies were made on tissues of cyclic hematopoietic (CH) dogs of various ages presenting a high incidence of spontaneous amyloidosis. The distribution and morphologic characteristics of amyloidosis in this animal model closely correspond to the secondary and familial forms of the disease in humans. Plasma cells and, particularly, macrophages presented marked changes during the evolution of amyloid deposition. Residual bodies in the macrophages contained abundant cell debris, a result of both endocytic and autophagocytic activities. Intracellular amyloid fibrils were not observed by conventional electron microscopy. A few reticular cells contained intracytoplasmic fibrils which were morphologically different from amyloid. There was no correlation between the amount of intracellular fibrils and the size of the extracellular amyloid deposits. On the contrary, a temporal association between the magnitude of the amyloid deposits and cytoplasmic changes in the macrophages at sequential stages of the evolution of the disease was evident. It is suggested that the hematopoietic defect in the CH dog could play an important role in the production of amyloidosis, making this animal an excellent experimental model for studies of that disease.Supported by Grants NIH RR00874, NIH HL 15647, and NIH FR5541 from the National Institutes of Health, DHEW     

Isolation and characterization of amyloid protein AA in the Abyssinian cat

Amyloid fibrils were isolated by extraction in deionized water from the kidneys of an Abyssinian cat with familial renal amyloidosis. The fibrils were suspended in a buffer containing 6 M guanidine hydrochloride and reduced and alkylated using dithiothreitol and iodoacetid acid. The resulting amyloid fibril subunit protein was isolated by chromatography on a column of Sepharose CL6B. It was fragmented using cyanogen bromide, and the resultant peptides were separated by reverse phase high performance liquid chromatography. The protein was characterized by determination of the amino acid sequence of the cyanogen bromide fragments using a Beckman 890C sequencer. The primary structure of this amyloid fibril subunit protein showed strong homology with amyloid protein AA found in man and animals with spontaneous and experimentally induced reactive systemic amyloidosis. This study confirms the reactive nature of familial renal amyloidosis in the Abyssinian cat and suggests that this disease may be a valuable spontaneous animal model for the study of familial Mediterranean fever in man.     

Fine structure and origin of amyloid deposits in pituitary adenoma.

A pituitary adenoma with amyloid deposits was studied by light and electron microscopy. The amyloid material was radially oriented, deposited in the extracellular space, and often intermingled with small vesicles. It was surrounded by histiocytes with deep cytoplasmic invaginations. The histiocytes contained large autophagic vacuoles in which amyloid was also present. These features are similar to other forms of amyloidosis and suggest a histiocytic origin of the amyloid.     

Amyloid in familial amyloidosis, Finnish type, is antigenically and structurally related to gelsolin.

Immunohistochemical studies of six patients with familial amyloidosis, Finnish type, showed that their amyloid deposits did not react with polyclonal antibodies against the amyloid proteins of other, established forms of systemic or cerebral amyloidosis. However, strong immunoreactivity was observed with rabbit antiserum raised against a low molecular weight purified amyloid subunit isolated from one of the patients. This immunoreactivity was abolished by absorption with the low molecular weight amyloid fraction. The amino terminal sequence of the amyloid protein subunit was homologous to gelsolin, an actin-modulating protein, and the amyloid deposits in tissues reacted with a monoclonal antibody against gelsolin. These studies show that the amyloid protein in familial amyloidosis, Finnish type, is not related to previously identified forms of amyloid, including prealbumin (transthyretin) variants, but represents a novel amyloidogenic protein related to gelsolin, a plasma and cytoplasmic protein.     

Relationship between amyloid deposition and intracellular structural changes in familial amyloidotic polyneuropathy

Transthyretin-related familial amyloidotic polyneuropathy is a systemic amyloidosis caused by mutations in the transthyretin gene. Extracellular deposition of amyloid is the common pathologic hallmark of amyloidoses including Alzheimer disease, AL amyloidosis, AA amyloidosis, and familial amyloidotic polyneuropathy. However, the exact relationship between amyloid deposition and cell death has not yet been clarified. To elucidate this relationship, we studied the effect of transthyretin amyloid fibrils and prefibrillar aggregates on cells by using autopsy tissues obtained from 8 patients with familial amyloidotic polyneuropathy, as well as cultured cell lines. Ultrastructural studies of amyloid-laden cardiomyocytes showed that intracellular structural changes correlated with the degree of amyloid deposition and may reflect metabolic disturbances caused by physical limitations imposed by the amyloid deposits. Amyloid-laden vascular endothelial cells, mesangial cells, smooth muscle cells, Schwann cells, and cardiomyocytes, however, had well-preserved cell nuclei and showed no apoptotic changes, even when cells were completely surrounded by prefibrillar transthyretin aggregates and amyloid fibrils. Synthesized prefibrillar transthyretin aggregates, transthyretin fibrils, and amyloid fibrils obtained from patients with familial amyloidotic polyneuropathy evidenced no cytotoxicity in cell culture experiments. Our data thus indicate that neither transthyretin amyloid fibrils nor prefibrillar transthyretin aggregates directly induced apoptosis. However, cellular metabolic disturbances caused by cells' being physically confined by amyloid deposits may induce cell degeneration.     

Amyloid goiter and arthritides after kidney transplantation in a patient with systemic amyloidosis and Muckle-Wells syndrome

A case of hereditary AA amyloidosis with Muckle-Wells syndrome is described. After a successful kidney transplantation for chronic renal failure due to renal amyloid deposits at age 21, the patient, a white female now 26 years of age, developed a large amyloid goiter as a manifestation of the systemic amyloidosis and recurrent monarthritides. Both observations are novel for this disease. Subtotal thyroidectomy and oral colchicine administration, known to be effective in preventing complications of familial Mediterranean fever, another hereditary type of AA amyloidosis, proved highly effective in the management of this unusual case.     

Nephrotic syndrome and renal insufficiency in association with amyloidosis: A correlation between structure and function

Summary The following results were obtained by correlating light and electron microscopic findings from 85 cases of glomerular renal amyloidosis with clinical parameters:1. Amyloid masses deposited in the glomeruli do not represent an effective filtration barrier, i.e., the formation of primary urine is not significantly influenced even by extensive amyloid masses in the glomeruli; protein retention is first observed when the glomerular capillaries are almost totally obliterated.2. Once the nephrotic syndrome has developed in association with glomerular renal amyloidosis, it shows no tendency for remittance, despite progressing renal insufficiency.3. The reason for this persistance of the nephrotic syndrome, despite increasing renal insufficiency, is a progressive reduction in the capacity of the tubules for protein reabsorption in the presence of increasing interstitial fibrosis of the kidney.4. Interstitial fibrosis of the kidney cortex leads to increasing impairment in the oxygen and energy supply to the tubule cells together with considerable functional deterioration which, in addition to other metabolic disturbances, also results in a reduced capacity for protein reabsorption.Supported by Deutsche Forschungsgemeinschaft     

A putative role for cathepsin K in degradation of AA and AL amyloidosis

The aims of this study were to investigate the role of cathepsin K in the pathology of amyloidosis by demonstrating its presence in multinucleated giant cells (MGCs) adjacent to amyloid deposits, and determining its ability to degrade amyloid fibril proteins in vitro. The study was performed using autopsy and biopsy specimens from patients with AA or AL amyloidosis. In six (55%) patients with AA amyloidosis and seven (58%) patients with AL amyloidosis, variable numbers of CD68-immunoreactive MGCs were found adjacent to amyloid deposits. In each case strong cytoplasmic immunostaining for cathepsin K was found in MGCs; immunostaining of amyloid deposits was present in five (45%) patients with AA amyloidosis and three (25%) patients with AL amyloidosis. In vitro degradation experiments showed that recombinant cathepsin K completely degraded AA amyloid fibril proteins at pH 5.5 as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Less effective degradation took place at pH 7.4 and there was no degradation in the presence of a general cysteine protease inhibitor (E64) or in the absence of cathepsin K. This is the first study to show that cathepsin K is expressed in MGCs adjacent to amyloid deposits and to demonstrate its ability to degrade amyloid fibril proteins.     

Pulmonary vascular amyloidosis in aged dogs. A new form of spontaneously occurring amyloidosis derived from apolipoprotein AI.

The N-terminus of a mutant form of apolipoprotein AI [apoAI] has previously been shown to be the subunit protein of amyloid fibrils in a human kindred with a form of familial amyloid polyneuropathy (FAP, type III) and in a recently reported kindred with a form of non-neuropathic hereditary amyloidosis. In this study, we demonstrate by amino-acid sequence analysis, that a form of vascular amyloidosis occurring in the lungs of aged dogs is derived from a N-terminal fragment of apoAI and that no amino acid substitution is present in this confirmed sequence. This represents the first documentation of apoAI as a precursor for a form of amyloidosis in animals, and provides the first documentation of apoAI as a precursor for amyloid fibrils in a form of age-associated ("senile") amyloidosis. Secondary structure prediction analysis of the N-terminal regions of normal human and dog apoAI indicated a propensity for beta-pleated sheet conformation, and thus amyloidogenesis, in 40 and 45% of the respective sequences. These results suggest that apoAI (like transthyretin) may serve as an amyloid precursor protein for both familial and senile forms of amyloidosis. ApoAI should, therefore, be considered as a potential amyloid precursor when forms of human senile amyloidosis of unknown origin are evaluated.     

Increased density of interstitial mast cells in amyloid A renal amyloidosis.

Renal interstitial fibrosis is the final common pathway leading to end-stage renal disease in various nephropathies including renal amyloidosis. However, the role of mast cells (MCs) in the fibrotic process of renal amyloidosis is not fully understood. We compared the distribution of MCs in renal biopsies from 30 patients with AA type renal amyloidosis and 20 control cases. Immunoreactivity of renal MCs to anti-tryptase and anti-chymase was studied. Interstitial myofibroblasts were stained with anti-alpha-smooth muscle actin (alpha-SMA) antibody, and inflammatory cells were identified by anti-CD45, -CD20, and -CD68 mAbs. Positively stained cells were counted, and the relative interstitial and fractional areas of anti-alpha-SMA stained cells were measured. Anti-CD29 mAb was used to detect beta1 integrin and anti-basic fibroblast growth factor (bFGF) mAb for the growth factor on MCs. MCs were rarely found in control samples. In contrast, samples showing amyloid deposition contained numerous tryptase-positive (MCT) (940.17 +/- 5.4 versus 6.74 +/- 1.1/mm2) but fewer chymase-positive (MCTC) cells (20.7 +/- 2.86 versus 1.7 +/- 0.76/mm2) in the renal interstitium. There was a significant relationship between interstitial MCT and creatinine clearance (r = -0.72), and between interstitial MCT and glomerular amyloid-index (GAI) (r = 0.723) and interstitial amyloid area (r = 0.824). Accumulation of MCs correlated significantly with the number of T lymphocytes (MCT: r = 0.694). There was also a significant relationship between mast cell (MC) number and the fractional area of alpha-SMA positive interstitium (r = 0.733) and interstitial fibrotic area (r = 0.6). Double immunostaining demonstrated intracytoplasmic presence of beta1 integrin on 87% of MCT and correlated significantly with the interstitial amyloid area (r = 0.818, P = .001) and T-cell number (r = 0.639, P = .002). bFGF was also detected on 85.5% of MCTC correlating well with the interstitial alpha-SMA-area (r = 0.789). Our results indicate that MCs constitute an integral part of the overall inflammatory process and play a crucial role in interstitial fibrosis in renal amyloidosis.     

Energetic characteristics of the new transthyretin variant A25T may explain its atypical central nervous system pathology

Transthyretin (TTR) is a tetrameric protein that must misfold to form amyloid fibrils. Misfolding includes rate-limiting tetramer dissociation, followed by fast tertiary structural changes that enable aggregation. Amyloidogenesis of wild-type (WT) TTR causes a late-onset cardiac disease called senile systemic amyloidosis. The aggregation of one of > 80 TTR variants leads to familial amyloidosis encompassing a collection of disorders characterized by peripheral neuropathy and/or cardiomyopathy. Prominent central nervous system (CNS) impairment is rare in TTR amyloidosis. Herein, we identify a new A25T TTR variant in a Japanese patient who presented with CNS amyloidosis at age 42 and peripheral neuropathy at age 44. The A25T variant is the most destabilized and fastest dissociating TTR tetramer published to date, yet, surprising, disease onset is in the fifth decade. Quantification of A25T TTR in the serum of this heterozygote reveals low levels relative to WT, suggesting that protein concentration influences disease phenotype. Another recently characterized TTR CNS variant (D18G TTR) exhibits strictly analogous characteristics, suggesting that instability coupled with low serum concentrations is the signature of CNS pathology and protects against early-onset systemic amyloidosis. The low A25T serum concentration may be explained either by impaired secretion from the liver or by increased clearance, both scenarios consistent with A25T's low kinetic and thermodynamic stability. Liver transplantation is the only known treatment for familial amyloid polyneuropathy. This is a form of gene therapy that removes the variant protein from serum preventing systemic amyloidosis. Unfortunately, the choroid plexus would have to be resected to remove A25T from the CSF-the source of the CNS TTR amyloid. Herein we demonstrate that small-molecule tetramer stabilizers represent an attractive therapeutic strategy to inhibit A25T misfolding and CNS amyloidosis. Specifically, 2-[(3,5-dichlorophenyl)amino]benzoic acid is an excellent inhibitor of A25T TTR amyloidosis in vitro.     

Deposition of Transthyretin in Early Stages of Familial Amyloidotic Polyneuropathy : Evidence for Toxicity of Nonfibrillar Aggregates

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. No systematic immunohistochemical data exists relating TTR deposition with FAP progression. We assessed nerves from FAP patients in different stages of disease progression (FAP 0 to FAP 3) for TTR deposition by immunohistochemistry, and for the presence of amyloid fibrils by Congo Red staining. The nature of the deposited material was further studied by electron microscopy. We observed that early in FAP (FAP 0), TTR is already deposited in an aggregated nonfibrillar form, negative by Congo Red staining. This suggested that in vivo, preamyloidogenic forms of TTR exist in the nerve, in a stage before fibril formation. Cytotoxicity of nonfibrillar TTR was assessed in nerves of different FAP stages by immunohistochemistry for macrophage colony-stimulating factor. FAP 0 patients already presented increased axonal expression of macrophage colony-stimulating factor that was maintained in all other stages, in sites related to TTR deposition. Toxicity of synthetic TTR fibrils formed in vitro at physiological pH was studied on a Schwannoma cell line by caspase-3 activation assays and showed that early aggregates but not mature fibrils are toxic to cells. Taken together, these results show that nonfibrillar cytotoxic deposits occur in early stages of FAP.