ODAM Expression Inhibits Human Breast Cancer Tumorigenesis.

We have posited that Odontogenic Ameloblast Associated Protein (ODAM) serves as a novel prognostic biomarker in breast cancer and now have investigated its potential role in regulating tumor growth and metastasis. Human breast cancer MDA-MB-231 cells were transfected with a recombinant ODAM plasmid construct (or, as a control, the plasmid vector alone). ODAM expression increased adhesion and apoptosis of the transfected MDA-MB-231 cells and suppressed their growth rate, migratory activity, and capability to invade extracellular matrix-coated membranes. Implantation of such cells into mouse mammary fat pads resulted in significantly smaller tumors than occurred in animals that received control cells; furthermore, ODAM-expressing cells, when injected intravenously into mice, failed to metastasize, whereas the control-transfected counterparts produced extensive lung lesions. Our finding that induction of ODAM expression in human breast cancer cells markedly inhibited their neoplastic properties provides further evidence for the regulatory role of this molecule in tumorigenesis and, consequently, is of potential clinical import.

Generation and characterization of anti-AA amyloid-specific monoclonal antibodies.

AA amyloidosis results from the pathologic deposition in the kidneys and other organs of fibrils composed of N-terminal fragments of serum amyloid A protein (SAA). Given that there are only limited means to visualize these deposits, we have developed a series of mAbs, 2A4, 7D8, and 8G9, that bind specifically with nanomolar affinity to a carboxy-terminal epitope generated following proteolysis of SAA that yields the predominant component of AA amyloid deposits. Notably, these antibodies do not recognize native SAA, they retain their immunoreactivity when radiolabeled with I-125 and, after injection into AA amyloidotic mice, localize, as evidenced by autoradiography and micro-single photon emission computed tomography imaging, to histologically confirmed areas of amyloid deposition; namely, spleen, liver, and pancreas. The results of our in vitro and in vivo studies demonstrate the AA fibril-selectivity of mAbs 2A4, 7D8, and 8G9 and warrant further investigation into their role as novel diagnostic agents for patients with AA amyloidosis.

Leukocyte chemotactic factor 2 (LECT2)-associated renal amyloidosis: a case series.

BACKGROUND: Renal amyloidosis is characterized by the pathologic deposition within glomeruli and/or interstitium of congophilic fibrils, most often composed of either immunoglobulin light chains or serum amyloid A-related protein and, less commonly, mutated forms of apolipoproteins AI or AII, lysozyme, fibrinogen, gelsolin, or transthyretin. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 10 patients with renal amyloidosis who had an amyloidogenic protein that was not identified using routine immunohistochemistry. OUTCOMES: Clinical, pathologic, biochemical, and genetic characteristics. MEASUREMENTS: Tandem mass spectrometry was used to analyze fibrils extracted from sections of formalin-fixed paraffin-embedded amyloid-containing kidney biopsy specimen blocks. RESULTS: Chemical analyses showed peptides corresponding to the carboxy-terminal portion of the leukocyte chemotactic factor 2 (LECT2) molecule. In addition, deposits were immunostained using an anti-human LECT2 monoclonal antibody. Plasma specimens were available from 2 individuals for whom LECT2 concentration in these samples was within the reference range. Additionally, in 4 of the cases analyzed at the molecular level, isolation of genomic DNA and polymerase chain reaction amplification of LECT2-encoding exons showed no mutations. However, all were homozygous for the G allele encoding valine at position 40 in the mature protein, a finding confirmed using restriction enzyme analysis of the polymorphic site. LIMITATIONS: Causality is not addressed. CONCLUSIONS: Based on our studies, we posit that LECT2-associated renal amyloidosis represents a unique and perhaps not uncommon disease, especially in Mexican Americans. The pathogenesis, extent, and prognosis remain to be determined.

Inhibition of pathologic immunoglobulin-free light chain production by small interfering RNA molecules.

OBJECTIVE: Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal immunoglobulin light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved antiplasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference. MATERIALS AND METHODS: SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the cytomegalovirus promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the American Type Culture Collection (Manassas, VA, USA). Both were treated with small interfering RNA directed specifically to the V, J, or C portions of the molecules and then analyzed by enzyme-linked immunosorbent assay, flow cytometry, and real-time polymerase chain reaction. RESULTS: Transfected cells were found to constitutively express detectable quantities of messenger RNA and protein Wil and, after exposure to small interfering RNAs, an ∼ 40% reduction in messenger RNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. CONCLUSIONS: Our results have shown that RNA interference can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma.

Radioimmunodetection of amyloid deposits in patients with AL amyloidosis.

Care of patients with AL amyloidosis currently is limited by the lack of objective means to document disease extent, as well as therapeutic options that expedite removal of pathologic deposits. To address these issues, we have initiated a Phase I Exploratory IND study to determine the biodistribution of the fibril-reactive, amyloidolytic murine IgG1 mAb 11-1F4 labeled with I-124. Patients were infused with less than 1 mg (∼ 74 MBq) of GMP-grade antibody and imaged by PET/CT scan 48 and 120 hours later. Among 9 of 18 subjects, there was striking uptake of the reagent in liver, lymph nodes, bone marrow, intestine, or, unexpectedly, spleen (but not kidneys or heart). Generally, positive or negative results correlated with those obtained immunohistochemically using diagnostic tissue biopsy specimens. Based on these findings, we posit that (124)I-mAb m11-1F4 can be used to identify AL candidates for passive immunotherapy using the chimeric form of the antibody.

Prevalence and morphology of leukocyte chemotactic factor 2-associated amyloid in renal biopsies.

Renal pathologists identify the protein component of renal amyloid deposits by immunohistochemistry using antibodies against known amyloidogenic proteins. The majority of amyloid cases can be categorized by a simple antibody panel that includes immunoglobulin light chains lambda and kappa, and serum amyloid A. In some instances, however, these reagents do not recognize materials that stain with Congo red or yield ambiguous staining results, thus creating a diagnostic dilemma. Chemical analysis of fibrils extracted from such a nonreactive renal biopsy led to the discovery of a previously unknown amyloid formed from leukocyte chemotactic factor 2 (LECT2). Over the past 8 years, we received 285 renal amyloid samples, of which 31 remained unclassified. In an effort to determine whether any of the latter samples were LECT2 related, tandem mass spectrometry was performed. In all, 7 of the 31 cases were identified as an amyloid LECT2 (ALECT2), a finding confirmed immunohistochemically using a LECT2-specific antibody. The deposits strongly stained for Congo red and, in most cases, had distinctive morphological features with diffuse involvement of the interstitium, arteries, and glomeruli. Hence, we believe that ALECT2 represents the third common form of renal amyloidosis.

Splenic plasma cells can serve as a source of amyloidogenic light chains.

Bone marrow-derived clonal plasma cells, as found in systemic amyloidogenic light chain-associated (AL) amyloidosis, are presumed to be the source of light chains that deposit as fibrils in tissues throughout the body. Paradoxically, people with this disorder, in contrast to multiple myeloma, often have a low percentage of such cells, and it is unknown whether this relatively sparse number can synthesize enough amyloidogenic precursor to form the extensive pathology that occurs. To investigate whether another hematopoietic organ, the spleen, also contains monoclonal light chain-producing plasma cells, we have immunostained such tissue from 26 AL patients with the use of antiplasma cell, antifree kappa and lambda, and anti-V(L) subgroup-specific monoclonal antibodies (mAbs). In 12 cases, there was statistically significant evidence of a monoclonal population bearing the same kappa or lambda isotype as that within the bone marrow and identical to the amyloid. Our studies have shown that the spleen may be another source of amyloidogenic light chains.

AA amyloidosis associated with a mutated serum amyloid A4 protein.

AA amyloidosis invariably has been associated with fibrillar deposits of the acute phase high-density lipoprotein serum amyloid A isotypes SAA1 and SAA2. We now report the first case in a patient with no antecedent history of a chronic inflammatory or neoplastic process whose pathologic renal deposits were comprised of a mutated form of the constitutively expressed serum amyloid A4 (SAA4) protein. Analyses by tandem mass spectrometry of amyloid extracted from kidney biopsies revealed a component identical in sequence to the N-terminal portion of SAA4, except for the substitution of glycine for tryptophan at position 22 (W22G). Sequencing of genomic DNA using SAA4-specific primers showed a TGG to GGG transversion in codon 22 that accounted for the observed modification. Confirmation of the SAA4 nature of the amyloid was obtained immunohistochemically. Notably, only wild-type SAA4 was detected by mass spectrometry in the patient's serum and its concentration was within normal limits. Given the substitution of an amino acid lacking a side chain for a bulky residue, we posit that the W22G alteration would profoundly affect SAA4 stability, rendering it amyloidogenic. Our studies provide the first evidence for a novel type of AA amyloidosis in which the fibrils were formed from a mutated SAA4 protein.

Immunodiagnostic capabilities of anti-free immunoglobulin light chain monoclonal antibodies.

Overproduction of plasma cell-derived monoclonal free kappa or lambda immunoglobulin light chains (FLCs) is a hallmark of multiple myeloma, AL amyloidosis, and light chain deposition disease. Because these components serve as unique cellular and serologic biomarkers, their detection and quantitation has diagnostic, therapeutic, and prognostic import. In this regard, we have developed monoclonal antibodies (mAbs) that specifically recognize the kappa or lambda FLC products of all known human variable and constant region light chain genes. We now report the results of our studies that have demonstrated the capability of these reagents to measure, in a modified fluid-phase capture enzyme-linked immunosorbent assay (ELISA), serum kappa and lambda FLCs at concentrations as low as 5 and 15 ng/mL, respectively. The mAb-based ELISA has greater sensitivity and reproducibility than does the commercially available immunoturbidimetric assay that uses polyclonal anti-FLC antibodies. In addition, the mAbs can immunostain monoclonal FLC-producing plasma cells and pathologic light chain-related amyloid and nonfibrillar tissue deposits. Our anti-FLC mAbs, with their high degree of reactivity and versatility, may provide an invaluable tool in the diagnosis and management of light chain-associated disease.

Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

Expression of odontogenic ameloblast-associated protein (ODAM) in dental and other epithelial neoplasms.

We previously have communicated our discovery that the amyloid associated with calcifying epithelial odontogenic tumors is composed of N-terminal fragments of the structurally novel odontogenic ameloblast-associated protein designated ODAM. Subsequently, it was shown by other investigators that ODAM is expressed in rodent enamel organ and is likely involved in dental development. We now report that this molecule also is found in certain human tissues, principally the salivary gland and trachea, as evidenced by RNA array analysis and immunohistochemistry-utilizing antibodies prepared against synthetic ODAM-related peptides and recombinant protein. Notably, these reagents immunostained normal and malignant ameloblasts and other types of human neoplastic cells, including those of gastric, lung, and breast origin where the presence in the latter was confirmed by in situ hybridization using gene-specific molecular probes. Moreover, significant titers of anti-ODAM IgG antibodies were detected in the sera of patients with these malignancies. Our studies have provided the first evidence in humans for the cellular expression of ODAM in normal and diseased states. Based on our findings, we posit that ODAM is a developmental antigen that has an essential role in tooth maturation and in the pathogenesis of certain odontogenic and other epithelial neoplasms; further, we suggest that ODAM may serve as a novel prognostic biomarker, as well as a potential diagnostic and therapeutic target for patients with breast and other epithelial forms of cancer.

Odontogenic ameloblast-associated protein nature of the amyloid found in calcifying epithelial odontogenic tumors and unerupted tooth follicles.

We have previously reported that the amyloid found in three patients with calcifying epithelial odontogenic tumors (CEOT) was composed of N-terminal fragments of a putative 153-residue protein specified by a gene designated FLJ20513 now known to represent exons 5 through 10 of the odontogenic ameloblast-associated protein (ODAM) locus that encodes a 279-residue polypeptide. Confirmation of the amyloidogenic potential of ODAM has resulted from analyses of four other cases where we found, in addition, a 74-residue segment specified by exon 4. Through preparation of ODAM-related synthetic peptides, it was possible to localize the fibril-forming region of this molecule, as well as generate a monoclonal antibody that reacted specifically with the amyloid associated with CEOT. Notably, we also detected green birefringent congophilic material in unerupted tooth follicles - a precursor of CEOT - and demonstrated through immunologic and chemical analyses the ODAM nature of the deposits. Our studies have provided further evidence for this unique form of odontogenic amyloid that we provisionally designate "AODAM".

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope.

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.

Amyloidogenic potential of foie gras.

The human cerebral and systemic amyloidoses and prion-associated spongiform encephalopathies are acquired or inherited protein folding disorders in which normally soluble proteins or peptides are converted into fibrillar aggregates. This is a nucleation-dependent process that can be initiated or accelerated by fibril seeds formed from homologous or heterologous amyloidogenic precursors that serve as an amyloid enhancing factor (AEF) and has pathogenic significance in that disease may be transmitted by oral ingestion or parenteral administration of these conformationally altered components. Except for infected brain tissue, specific dietary sources of AEF have not been identified. Here we report that commercially available duck- or goose-derived foie gras contains birefringent congophilic fibrillar material composed of serum amyloid A-related protein that acted as a potent AEF in a transgenic murine model of secondary (amyloid A protein) amyloidosis. When such mice were injected with or fed amyloid extracted from foie gras, the animals developed extensive systemic pathological deposits. These experimental data provide evidence that an amyloid-containing food product hastened the development of amyloid protein A amyloidosis in a susceptible population. On this basis, we posit that this and perhaps other forms of amyloidosis may be transmissible, akin to the infectious nature of prion-related illnesses.

Localization of a conformational epitope common to non-native and fibrillar immunoglobulin light chains.

Amyloid fibrils and partially unfolded intermediates may be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we had previously reported that the IgG1 mAb 11-1F4, generated by immunizing mice with a thermally denatured variable region fragment of the human Igkappa4 Bence Jones protein Len, reacted specifically with light chain (LC) fibrils, irrespective of kappa or lambda isotype but, notably, did not with native molecules (Hrncic, R. et al. (2000) Am. J. Pathol. 157, 1239-1246). To elucidate the molecular basis of this specificity, we have used a europium-linked fluorescent immunoassay, where it was demonstrated through epitope mapping that mAb 11-1F4 recognizes a conformational determinant contained within the first (N-terminal) 18 amino acids of misfolded LCs. The nature of this epitope was evidenced in competition studies where the peptide Len (1-18), but not the intact protein or other LCs, inhibited the binding of the antibody to fibrils. This unique reactivity was dependent on the structural integrity of this portion of the molecule, particularly the presence of a highly conserved prolyl residue at position 8. On the basis of our experimental data, we posit that the mAb 11-1F4 binding site found on partially denatured and fibrillar LCs involves an inducible N-terminal main chain reversal that results in the formation of a proline anchored beta-turn. Our delineation of this LC fibril-associated epitope provides the rationale for the design of novel amyloid-reactive antibodies with diagnostic and therapeutic potential for patients with LC-associated and other forms of amyloidosis.

Radioimaging of light chain amyloid with a fibril-reactive monoclonal antibody.

UNLABELLED: Currently, there are no available means in the United States to document objectively the location and extent of amyloid deposits in patients with systemic forms of amyloidosis. To address this limitation, we have developed a novel diagnostic strategy, namely, the use of a radiolabeled fibril-reactive murine monoclonal antibody (mAb) as an amyloid-specific imaging agent. The goal of this study was to determine the pharmacokinetics, biodistribution, and ability of this reagent to target the type of amyloid that is formed from immunoglobulin light chains, that is, AL. METHODS: Subcutaneous tumors (amyloidomas) were induced in BALB/c mice by injection of human AL fibrils. The IgG1 mAb designated 11-1F4 and an isotype-matched control antibody were radioiodinated, and the pharmacokinetics and localization of these reagents were determined from blood and tissue samples. Amyloidoma-bearing animals that received (125)I- or (124)I-labeled antibodies were imaged by whole-body small-animal SPECT/CT or small-animal PET/CT technology, respectively. RESULTS: Radioiodinated mAb 11-1F4 retained immunoreactivity, as evidenced by its subnanomolar affinity for light chains immobilized on 96-well microtiter plates and for beads conjugated with a light chain-related peptide. Additionally, after intravenous administration, the labeled reagents had the expected biologic half-life of murine IgG1, with monoexponential whole-body clearance kinetics. In the amyloidoma mouse model, (125)I-11-1F4 was predominately localized in the tumors, as demonstrated in biodistribution and autoradiographic analyses. The mean uptake of this reagent, that is, the percentage injected dose per gram of tissue, 72 h after injection was significantly higher for amyloid than for skeletal muscle, spleen, kidney, heart, liver, or other tissue samples. Notably, the accumulation within the amyloidomas of (125)I- or (124)I-11-1F4 was readily visible in the fused small-animal SPECT/CT or small-animal PET/CT images, respectively. CONCLUSION: Our studies demonstrate the amyloid-imaging capability of a radiolabeled fibril-reactive mAb and provide the basis for a clinical trial designed to determine its diagnostic potential in patients with AL amyloidosis and other systemic amyloidoses.

Amyloid contained in the knee joint meniscus is formed from apolipoprotein A-I.

OBJECTIVE: To determine the chemical nature of amyloid deposits found in knee joint menisci. METHODS: Amyloid was extracted from the menisci of 3 adults who underwent knee joint replacement surgery. The primary structural features of the purified proteins were determined by sequential Edman degradation and tandem mass spectrometry (MS/MS). Tissue specimens were also subjected to in situ hybridization analysis, as well as complementary DNA cloning by reverse transcriptase-polymerase chain reaction (RT-PCR). Additionally, specimens from these 3 patients, as well as other patients with amyloid in the knee joint menisci, were examined immunohistochemically. RESULTS: Amino acid sequence and MS/MS analyses of the extracts revealed the presence of 60-77-residue components identical to the N-terminal portion of apolipoprotein A-I (Apo A-I). The Apo A-I nature of the amyloid was confirmed by the demonstration that the green birefringent congophilic deposits in the 7 meniscus samples were recognized by an anti-human Apo A-I antibody. That the meniscus itself was the source of the amyloidogenic protein was evidenced through Southern blot analysis, in which an Apo A-I product was generated by RT-PCR from synovial tissue, and further, by the demonstration that the cytoplasm of chondrocytes reacted with the specific Apo A-I probe used for in situ hybridization and was immunostained by the anti-Apo A-I antiserum. CONCLUSION: Amyloid in the knee joint menisci is formed from Apo A-I that is produced by chondrocytes within the meniscal cartilage. This entity represents yet another localized form of amyloidosis associated with the aging process and may be of pathophysiologic import.

Characterization of systemic amyloid deposits by mass spectrometry.

The human systemic (noncerebral) amyloidoses represent a heterogeneous group of disorders characterized by the widespread deposition of proteins as fibrils in organs or tissues throughout the body. The unequivocal identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these illnesses. Heretofore, this information was inferred from clinical data, ancillary laboratory tests, and results of immunohistochemical, as well as genetic, analyses. However, to establish definitively the type of amyloid present, the chemical composition of the fibrillar components must be determined. For this purpose, we have developed micro-methods, whereby this information can be obtained by tandem mass spectrometry (MS/MS) using material extracted from formalin-fixed, amyloid-containing tissue biopsy specimens or subcutaneous fat aspirates. The ability to identify precisely the protein nature of the pathologic deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disease.

Diagnostic and therapeutic potential of amyloid-reactive IgG antibodies contained in human sera.

Passive immunotherapy using fibril-reactive mAbs has been shown experimentally to reduce amyloid formation and also accelerate amyloidolysis. We now report that human sera, as well as various sources of pooled human IgG, including pharmacologic formulations of immune globulin i.v. (IGIV), contain Abs that specifically recognize fibrils formed from light chains and other amyloidogenic precursor proteins, including serum amyloid A, transthyretin, islet amyloid polypeptide, and amyloid beta 1-40 peptide, but notably, do not react with these molecules in their native nonfibrillar forms. After isolation of the Abs from IGIV via fibril-conjugated affinity column chromatography, the EC50-binding value for light chains and amyloid beta 1-40 peptide fibrils was approximately 15 nM-a magnitude approximately 200 and 70 times less than that of the unbound fraction and unfractionated product, respectively. Comparable reactivity was found in the case of those formed from serum amyloid A, transthyretin, and islet amyloid polypeptide. The purified Abs immunostained human amyloid tissue deposits and could inhibit fibrillogenesis, as shown in fibril formation and extension assays. Most importantly, in vivo reactivity was evidenced in a murine model when the enriched Abs were used to image amyloid, as well as expedite its removal. These promising experimental results suggest that fibril affinity-purified IGIV has potential as a diagnostic and therapeutic agent for patients with amyloid-associated disease.

Quantitative high-resolution microradiographic imaging of amyloid deposits in a novel murine model of AA amyloidosis.

The mouse model of experimentally induced systemic AA amyloidosis is long established, well validated, and closely analogous to the human form of this disease. However, the induction of amyloid by experimental inflammation is unpredictable, inconsistent, and difficult to modulate. We have previously shown that murine AA amyloid deposits can be imaged using iodine-123 labeled SAP scintigraphy and report here substantial refinements in both the imaging technology and the mouse model itself. In this regard, we have generated a novel prototype of AA amyloid in which mice expressing the human interleukin 6 gene, when given amyloid enhancing factor, develop extensive and progressive systemic AA deposition without an inflammatory stimulus, i.e., a transgenic rapidly inducible amyloid disease (TRIAD) mouse. Additionally, we have constructed high-resolution micro single photon emission computed tomography (SPECT)/computed tomography (CT) instrumentation that provides images revealing the precise anatomic location of amyloid deposits labeled by radioiodinated serum amyloid P component (SAP). Based on reconstructed microSPECT/CT images, as well as autoradiographic, isotope biodistribution, and quantitative histochemical analyses, the (125)I-labeled SAP tracer bound specifically to hepatic and splenic amyloid in the TRIAD animals. The ability to discern radiographically the extent of amyloid burden in the TRIAD model provides a unique opportunity to evaluate the therapeutic efficacy of pharmacologic compounds designed to inhibit fibril formation or effect amyloid resolution.