Amyloid Enhancing Factor Research Articles

Studies on the potential risk of amyloidosis from exposure to cultured fibril from silk fibroin

Amyloid A (AA) amyloidosis is induced by administering amyloid fibrils to animals under inflammatory conditions. Silk fibroin (SF), the main component of silk threads, forms amyloid-like fibrils and has been previously reported to induce AA amyloidosis in mice. In this study, SF was cultured in ethanol solution, and after confirming fibril formation through thioflavin T assay, Congo red assay, and observation under electron microscopy, cultured SF ethanol solutions were administered to mice via various routes to investigate the induction of target organs and amyloidosis. As a result, cultured SF ethanol solutions were confirmed to reach the lungs and spleen, but no amyloid deposition was observed. While SF forms amyloid-like fibril structures through cultivation in ethanol solution, its amyloid-enhancing factor (AEF) activity is considered low in mice.

Chemical design of radioiodinated probes with a metabolizable linkage for target-selective imaging of systemic amyloidosis

IntroductionSystemic amyloidosis is a rare disease caused by the deposition of amyloid fibrils in various organs. Amyloid-targeted radiopharmaceuticals have been developed and applied to diagnose systemic amyloidosis peripherally; however, high-contrast imaging has not been achieved because of the high background signals in normal organs. To overcome this problem, we designed an amyloid-targeted radioiodinated probe 1 with a metabolizable linkage (ester bond) to release of radiolabeled metabolites (m-iodohippuric acid) in normal organs that could be rapidly excreted in the urine. MethodsCompound 1 was synthesized by conjugating 2-(4-(methylamino)phenyl)benzo[d]thiazol-6-ol, an amyloid-targeting compound, with m-iodohippuric acid. [125I]1 was synthesized via iododestannylation using a tributyltin precursor. Mouse models of amyloid A (AA) amyloidosis, a type of systemic amyloidosis, were prepared by administering amyloid-enhancing factor to mice and used for in vitro autoradiography using organ sections and in vivo evaluation. Results[125I]1 was obtained with a radiochemical yield of 59% and radiochemical purity of over 95%. An in vitro autoradiographic study demonstrated that [125I]1 specifically binds to amyloid in the splenic tissue. Upon administration to normal mice, [125I]1 was distributed to organs throughout the body, followed by the rapid excretion of radioactivity in the urine as m-[125I]iodohippuric acid. Furthermore, ex vivo autoradiography showed that [125I]1 bound to the amyloid formed around the follicles in the spleens of AA amyloidosis model mice. ConclusionThese results suggest that the interposition of a metabolizable linkage between an amyloid-targeting moiety and a radiolabeled hippuric acid would be useful in the design of radiopharmaceuticals for high-contrast imaging of systemic amyloidosis.

Local administration of amyloid enhancing factor initiates in situ amyloid A deposition followed by systemic lesions in mice.

Amyloid A (AA) amyloidosis is experimentally transmissible in some animal species, such as mice and chickens. While the spleen is important as the initial deposition site in the transmission of AA amyloidosis, it is not essential for establishing the transmission, and its role is not precisely understood. In this study, to clarify why the spleen is the first site of deposition in transmissible AA amyloidosis, we administered amyloid enhancing factor, which is AA fibrils extracted from AA amyloidosis affected mouse to local organs (liver, spleen, kidney, stomach wall, and Peyer's patches), to tail vein and into peritoneum; then compared the amyloid distribution. Interestingly, initial amyloid deposition was observed at the administration site in each administered organ, not just the spleen. Furthermore, the amount of amyloid deposition in intra-organ administration groups was larger than that of the intravenous or intraperitoneal administration groups. This study indicates that locally exposed AEF initiates in situ amyloid deposition, from which amyloid deposition spreads throughout the body.

Development of a radioiodinated thioflavin-T-Congo-red hybrid probe for diagnosis of systemic amyloidosis

IntroductionSystemic amyloidosis is a group of diseases characterized by the deposition of amyloid protein in multiple organs throughout the body and causing their dysfunction. As amyloid deposition is observed at an early phase and is highly specific to systemic amyloidosis, noninvasive detection of amyloid is considered useful for the early diagnosis of systemic amyloidosis. In this study, we designed and synthesized a novel radiolabeled amyloid imaging probe, sodium (E)-4-amino-3-((4-(6-iodobenzothiazol-2-yl)phenyl)diazenyl)naphthalene-1-sulfonate (1), which combines two amyloid-binding compounds, thioflavin-T and Congo-red, and evaluated its effectiveness in diagnosing amyloidosis. MethodsA tributyltin precursor was synthesized through a 5-step reaction from 2-amino-6-bromobenzothiazole, and [125I]1 was synthesized by an iododestannylation reaction with a tributyltin precursor. Mouse models of amyloid A (AA) amyloidosis, a type of systemic amyloidosis, were prepared by intraperitoneal injection of amyloid-enhancing factor into mice. An in vitro autoradiographic study was performed using spleen sections from normal mice and AA amyloidosis mice. Furthermore, [125I]1 was intravenously injected into mice, and its distribution was evaluated. Finally, an ex vivo autoradiographic study was performed using AA amyloidosis mice. Results[125I]1 was obtained with a radiochemical yield of 66% and a radiochemical purity of over 95%. In vitro autoradiography revealed specific binding of [125I]1 to thioflavin-S-stained regions in the spleen. Normal mice showed relatively rapid clearance of [125I]1 from the organs, whereas radioactivity was retained in the spleen, where amyloid deposition was observed in model mice. Furthermore, ex vivo autoradiography showed a heterogeneous distribution of [125I]1, which was co-localized with thioflavin-S-stained regions in the spleen of model mice. ConclusionThese results indicate the potential of radioiodinated 1 as a nuclear imaging probe for diagnosing AA amyloidosis.

Aggregation of Mouse Serum Amyloid A Protein Was Promoted by Amyloid-Enhancing Factors with the More Genetically Homologous Serum Amyloid A

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-β structure accumulate and are deposited extracellularly in organs, resulting in chronic inflammatory diseases and infections. The incidence of AA amyloidosis is high in humans and several animal species. Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis. Amyloid enhancing factor (AEF) serves as a seed for fibril formation and shortens the onset of AA amyloidosis sharply. In this study, we examined whether AEFs extracted and purified from five animal species (camel, cat, cattle, goat, and mouse) could promote mouse SAA (mSAA) protein aggregation in vitro using quantum-dot (QD) nanoprobes to visualize the aggregation. The results showed that AEFs shortened and promoted mSAA aggregation. In addition, mouse and cat AEFs showed higher mSAA aggregation-promoting activity than the camel, cattle, and goat AEFs. Interestingly, homology analysis of SAA in these five animal species revealed a more similar amino acid sequence homology between mouse and cat than between other animal species. Furthermore, a detailed comparison of amino acid sequences suggested that it was important to mSAA aggregation-promoting activity that the 48th amino acid was a basic residue (Lys) and the 125th amino acid was an acidic residue (Asp or Glu). These data imply that AA amyloidosis exhibits higher transmission activity among animals carrying genetically homologous SAA gene, and may provide a new understanding of the pathogenesis of amyloidosis.

Lipid membranes accelerate amyloid formation in the mouse model of AA amyloidosis

Introduction: AA amyloidosis develops as a result of prolonged inflammation and is characterized by deposits of N-terminal proteolytic fragments of the acute phase reactant serum amyloid A (SAA). Macrophages are usually found adjacent to amyloid, suggesting their involvement in the formation and/or degradation of the amyloid fibrils. Furthermore, accumulating evidence suggests that lipid membranes accelerate the fibrillation of different amyloid proteins.Methods: Using an experimental mouse model of AA amyloidosis, we compared the amyloidogenic effect of liposomes and/or amyloid-enhancing factor (AEF). Inflammation was induced by subcutaneous injection of silver nitrate followed by intravenous injection of liposomes and/or AEF to accelerate amyloid formation.Results: We showed that liposomes accelerate amyloid formation in inflamed mice, but the amyloidogenic effect of liposomes was weaker compared with AEF. Regardless of the induction method, amyloid deposits were mainly found in the marginal zones of the spleen and coincided with the depletion of marginal zone macrophages, while red pulp macrophages and metallophilic marginal zone macrophages proved insensitive to amyloid deposition.Conclusions: We conclude that increased intracellular lipid content facilitates AA amyloid fibril formation and show that the mouse model of AA amyloidosis is a suitable system for further mechanistic studies.

Spontaneous, Experimentally Induced, and Transmissible AA Amyloidosis in Japanese Quail ( Coturnix japonica).

The authors describe a spontaneous case of amyloid A (AA) amyloidosis in an adult female Japanese quail ( Coturnix japonica). The bird developed AA amyloidosis secondary to chronic peritonitis caused by a Gram-negative bacillus infection. Mild amyloid deposition was also identified in the intestinal tract of apparently healthy adult individuals, suggesting that quail may develop intestinal amyloidosis with age. Based on these observations, it was hypothesized that quail can develop AA amyloidosis following inflammatory stimulation with lipopolysaccharide (LPS). Therefore, adult quail were repeatedly injected with LPS and the development of AA amyloidosis was confirmed. The amyloid deposition in this model increased when quail amyloid was intravenously injected as an amyloid-enhancing factor. The experiments were repeated with young quail, but amyloid deposits were not observed following LPS injections. However, AA amyloidosis did develop when quail amyloid was injected in addition to LPS. These results indicated that adult quail develop AA amyloidosis after inflammatory stimulation with LPS. Furthermore, quail AA amyloidosis was shown to have transmissibility regardless of age. Interestingly, the authors found that administration of chicken amyloid fibrils also induced AA amyloidosis in young quail. This is the first report of cross-species transmission of avian AA amyloidosis.

Deposition, Clearance, and Reinduction of Amyloid A Amyloid in Interleukin 1 Receptor Antagonist Knockout Mice.

Amyloid A (AA) amyloidosis is characterized by the extracellular deposition of AA amyloid and results in the irreversible dysfunction of parenchymal organs. In experimental models, AA amyloid deposits are cleared following a decrease in circulating serum amyloid A (SAA) concentrations. Additional inflammatory stimuli during this recovery process may induce more severe amyloid redeposition. In the present study, we confirmed the deposition, clearance, and reinduction of AA amyloid deposits in interleukin 1 receptor antagonist knockout mice (IL-1raKO) and studied the SAA levels and amyloid-enhancing factor activity based on the time-dependent changes of amyloid deposition. Histopathologically, following initial (day 0) injection of amyloid-enhancing factor in combination with an inflammatory stimulus (silver nitrate [AgNO3]), amyloid deposition peaked by day 20, and its deposition gradually decreased after day 35. SAA concentrations in serum were precipitously elevated on day 1 but returned to normal levels by day 10, whereas the SAA dimer was detected in serum after day 45. An additional AgNO3 injection was administered to mice with amyloidosis on day 5, 10, 35, or 50, and all mice developed large amyloid deposits. Amyloid deposition was most severe in mice treated with AgNO3 on day 35. The inoculation of sera from mice with AA amyloidosis, combined with AgNO3, induced AA amyloidosis. Serum samples collected on days 35 and 50, which contained high concentrations of the SAA dimer, induced amyloidosis in a high proportion (83%) of mice. Therefore, increased SAA and/or its dimer in serum during the recovery process may markedly exacerbate the development of AA amyloidosis.

Amyloid persistence in decellularized liver: biochemical and histopathological characterization

Systemic amyloidoses are a group of debilitating and often fatal diseases in which fibrillar protein aggregates are deposited in the extracellular spaces of a range of tissues. The molecular basis of amyloid formation and tissue localization is still unclear. Although it is likely that the extracellular matrix (ECM) plays an important role in amyloid deposition, this interaction is largely unexplored, mostly because current analytical approaches may alter the delicate and complicated three-dimensional architecture of both ECM and amyloid. We describe here a decellularization procedure for the amyloidotic mouse liver which allows high-resolution visualization of the interactions between amyloid and the constitutive fibers of the extracellular matrix. The primary structure of the fibrillar proteins remains intact and the amyloid fibrils retain their amyloid enhancing factor activity.

Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation after disappearance of amyloid depositions in mice.

Amyloid A (AA) amyloidosis is characterized by extracellular pathogenic deposition of insoluble fibril protein in various body organs. Deposited amyloid generally remains in a variety of organs for long periods, but its disappearance has been reported after the precursor protein is diminished. The kinetics of AA deposition are not completely understood and, in particular, the roles of cells and cytokines in the deposition and clearance of amyloid remain unclear. In this study, we investigated the disappearance of amyloid depositions in mice over a 1-year period. AA amyloidosis was induced experimentally in mice by injecting amyloid-enhancing factor (AEF) and silver nitrate. Mice were killed at different time-points to examine the occurrence and disappearance of amyloid depositions. Maximum levels of amyloid depositions were observed at 20 days after inoculation. Clearance of amyloid depositions was observed from the 40th day onwards, with only minute traces of amyloid present by 240 days. A second inflammatory stimulus consisting of AEF and silver nitrate was given at 330 or 430 days, after amyloid depositions had disappeared almost completely. After that, serum amyloid A was overproduced and redeposition of amyloid was observed, indicating that all mice were primed for aggressive amyloid depositions. After administration of the inflammatory stimuli, the proinflammatory environment was found to have increased levels of interleukin (IL)-6, while anti-inflammatory conditions were established by IL-10 as regression of amyloid deposition occurred. These results suggest that the proinflammatory and anti-inflammatory status have key roles in both amyloid deposition and clearance.

Phagocyte depletion inhibits AA amyloid accumulation in AEF-induced huIL-6 transgenic mice

Objective: Determine the role of phagocytosis in the deposition of acute phase SAA protein in peripheral organs as AA amyloid.Methods: AA amyloidosis was induced by injection of amyloid enhancing factor (AEF) in huIL-6 transgenic mice. Clodronate liposomes were injected at different times, and the amyloid load evaluated by Congo red birefringence staining and monitoring with the amyloid specific probe 125I-labeled peptide p5R.Results: Injection of clodronate containing liposomes depleted Iba-1 positive and F4/80 positive phagocytic cells in liver and spleen for up to 5 days. Treatment prior to administration of intravenous AEF did not alter the pattern of deposition of the AEF in spleen, but inhibited the catabolism of the 125I-labeled AEF. Clodronate treatment 1 day before or 1 day after AEF administration had little effect on AA amyloid accumulation at 2 weeks; however, mice treated with clodronate liposomes 5 days after AEF induction and evaluated at 2 weeks post-AEF induction showed reduced amyloid load relative to controls. At 6 weeks post-AEF there was no significant effect on amyloid load following a single clodronate treatment.Conclusion: Macrophages have been shown to be instrumental in both accumulation and clearance of AA amyloid after cessation of inflammation. Our data indicate that when SAA protein is continuously present, depletion of phagocytic cells during the early course of the disease progression temporarily reduces amyloid load.

Depletion of Spleen Macrophages Delays AA Amyloid Development: A Study Performed in the Rapid Mouse Model of AA Amyloidosis

AA amyloidosis is a systemic disease that develops secondary to chronic inflammatory diseases Macrophages are often found in the vicinity of amyloid deposits and considered to play a role in both formation and degradation of amyloid fibrils. In spleen reside at least three types of macrophages, red pulp macrophages (RPM), marginal zone macrophages (MZM), metallophilic marginal zone macrophages (MMZM). MMZM and MZM are located in the marginal zone and express a unique collection of scavenger receptors that are involved in the uptake of blood-born particles. The murine AA amyloid model that resembles the human form of the disease has been used to study amyloid effects on different macrophage populations. Amyloid was induced by intravenous injection of amyloid enhancing factor and subcutaneous injections of silver nitrate and macrophages were identified with specific antibodies. We show that MZMs are highly sensitive to amyloid and decrease in number progressively with increasing amyloid load. Total area of MMZMs is unaffected by amyloid but cells are activated and migrate into the white pulp. In a group of mice spleen macrophages were depleted by an intravenous injection of clodronate filled liposomes. Subsequent injections of AEF and silver nitrate showed a sustained amyloid development. RPMs that constitute the majority of macrophages in spleen, appear insensitive to amyloid and do not participate in amyloid formation.

Human SAA1-derived amyloid deposition in cell culture: a consistent model utilizing human peripheral blood mononuclear cells and serum-free medium

Amyloid A (AA) amyloidosis is a fatal disease caused by extracellular deposition of fibrils derived from serum AA (SAA). AA amyloid fibril formation has previously been modeled in macrophage cultures using highly amyloidogenic mouse SAA1.1, but attempts to do the same with human SAA invariably failed. Our objective was to define conditions that support human SAA-derived amyloid formation in peripheral blood mononuclear cell (PBMC) cultures. Two conditions were found to be critical – omission of fetal calf serum and use of StemPro34, a lipid-enriched medium formulated for hematopoietic progenitor cells. Cultures maintained in serum-free StemPro34 and provided with recombinant human SAA1 in the complete absence of amyloid-enhancing factor exhibited amyloid deposition within 7 d. Amyloid co-localized with cell clusters that characteristically included cells of fibrocytic/dendritic morphology as well as macrophages. These cells formed networks that appeared to serve as scaffolding within and upon which amyloid accumulated. Cells in amyloid-forming cultures demonstrated increased adherence, survival and expression of extracellular matrix components. Of the three human SAA1 isoforms, SAA1.3 showed the most extensive amyloid deposition, consistent with it being the most prevalent isoform in Japanese patients with AA amyloidosis. Attesting to the reproducibility and general applicability of this model, amyloid formation has been documented in cultures established from eight PBMC donors.

A novel method for quantifying peripheral tissue amyloid load by using the radiolabeled amyloidophilic peptide, p5

Quantitation of peripheral amyloid deposits by non-invasive molecular imaging can be useful for diagnosis, prognostication and monitoring response to therapy. In order to obtain reliable quantitative data, it is necessary to show a linear positive correlation between the uptake of the molecular probe and the tissue amyloid load. The transgenic H-2/IL-6 mouse model of AA amyloidosis was used to generate animals with varied stages of visceral amyloid disease. The mice were injected with 125I-labeled peptide p5 and tissues analyzed 2 h post-injection using Congo red (CR) staining, radioisotope biodistribution and micro-autoradiography (ARG). Micro-ARG confirmed that 125I-p5 was deposited at all amyloid deposits and sites of Congophilia but not at amyloid-free sites within the tissues evaluated. Furthermore, biodistribution studies revealed that the amount of 125I deposited in liver and spleen correlated with the amount of CR birefringence (expressed as 0–4+ or as tissue area [µm2]) in these tissues with correlation coefficients of r > 0.7 (p < 10−6). Deposition of 125I-p5 is a quantitative measure of the amount of AA amyloid in liver and spleen in this mouse model. The p5 peptide has potential as a quantitative amyloid imaging agent in human disease.Abbreviations: AEF, amyloid enhancing factor; CR, Congo red; HSPG, heparan sulfate proteoglycan; PET, positron emission tomography; SAA, serum amyloid protein A; SPECT, single photon emission computed tomography

AA-Amyloid is cleared by endogenous immunological mechanisms

Objective: AA amyloidosis is a complication to longstanding inflammatory diseases, but reduction of amyloid mass has been reported as the inflammation ceases. Not much is known about the endogenous factors that contribute to this amyloid resolution. Herein, we describe the dynamics of amyloid degradation and resolution in experimental murine AA-amyloidosis. Methods: AA-amyloidosis was induced in mice with injections of amyloid enhancing factor (AEF) and by inflammation induced with injections of silver nitrate. Resolution of amyloid deposits was monitored over time. Results: Virtually all amyloid was cleared within 34 weeks. Using the ELISA-technique, antibodies directed against protein AA were detected in animals during amyloid clearance phase and macrophages were shown to internalize amyloid. Also, passive immunization with an amyloid specific monoclonal antibody, produced by a B-cell clone recovered from an animal with advanced AA-amyloidosis, reduced amyloid development in murine AA-amyloidosis. Conclusion: Immunoglobulins co-localize with amyloid deposits and can contribute to amyloid degradation by Fc-receptor mediated phagocytosis, and should be considered key players in the degradation process.

Attempts at suppression of amyloidogenesis in a mouse model by a variety of anti-inflammatory agents

ObjectiveThe mainstay of AA amyloidosis prevention and treatment is suppression of inflammation. In the present study we have tried to determine the efficacy of a variety of anti-inflammatory agents at suppressing AA amyloidosis in a mouse model of the disease. MethodsAA amyloidosis was induced in Swiss male mice using amyloid enhancing factor and AgNO3. Suppression of amyloid formation was studied in comparison to saline, using i.p. injections of several non-steroidal anti-inflammatory agents, TNF-α inhibitors, interferon-α, leflunomide and a variety of chemotherapeutic agents, commonly used in the treatment of inflammatory illnesses such as methotrexate, azathioprine, chlorambucil and cyclophosphamide. The degree of splenic amyloid deposition was determined using Congo red staining of smears and a 5 grade scale. ResultsThe alkylating agents, chlorambucil and cyclophosphamide, each resulted in a significant 88% reduction in amyloid deposition, yielded the most striking effect on amyloidogenesis suppression in the enhanced mouse model (p<0.0002). The non-steroidal anti-inflammatory agents tested varied widely in their ability to suppress amyloid formation in our mouse model, but only diflunisal was significantly effective, inducing a suppression of 57% (p=0.04). Other chemotherapeutic agents tested, methotrexate and azathioprine, yielded 32% and 27% suppression, which fell short of statistical significance. Surprisingly, the immunomodulatory agents etanercept, infliximab, leflunomide and interferon-α had insignificant effects on amyloid formation in this model. ConclusionOur findings suggest that alkylating agents may have a role in the prevention of amyloidogenesis. Further testing of these agents in animal models and in the clinical setting is needed.

Accelerated Resolution of AA Amyloid in Heparanase Knockout Mice Is Associated with Matrix Metalloproteases

AA-amyloidosis is a disease characterized by abnormal deposition of serum A amyloid (SAA) peptide along with other components in various organs. The disease is a complication of inflammatory conditions that cause persistent high levels of the acute phase reactant SAA in plasma. In experimental animal models, the deposited amyloid is resolved when the inflammation is stopped, suggesting that there is an efficient clearance mechanism for the amyloid. As heparan sulfate (HS) is one of the major components in the amyloid, its metabolism is expected to affect the pathology of AA amyloidosis. In this study, we investigated the effect of heparanase, a HS degradation enzyme, in resolution of the AA amyloid. The transgenic mice deficient in heparanase (Hpa-KO) produced a similar level of SAA in plasma as the wildtype control (Ctr) mice upon induction by injection of AEF (amyloid enhancing factor) and inflammatory stimuli. The induction resulted in formation of SAA amyloid 7-days post treatment in the spleen that displayed a comparable degree of amyloid load in both groups. The amyloid became significantly less in the Hpa-KO spleen than in the Ctr spleen 10-days post treatment, and was completely resolved in the Hpa-KO spleen on day 21 post induction, while a substantial amount was still detected in the Ctr spleen. The rapid clearance of the amyloid in the Hpa-KO mice can be ascribed to upregulated matrix metalloproteases (MMPs) that are believed to contribute to degradation of the protein components in the AA amyloid. The results indicate that both heparanase and MMPs play important parts in the pathological process of AA amyloidosis.

Antisense oligonucleotide suppression of serum amyloid A reduces amyloid deposition in mice with AA amyloidosis

AA amyloid patients who experience disease progression and develop renal failure have not received sufficient benefit from agents that treat inflammation or infection. We have begun to explore the potential application of antisense oligonucleotides (ASOs) to specifically suppress SAA production and thereby reduce amyloid deposition. Proof-of-concept experiments conducted in mice initially examined ASO ability to reduce serum levels of SAA during an acute inflammatory response. Peak SAA levels in ASO-treated mice were reduced as much as 65% relative to levels in saline-treated mice. The extent of suppression was dose-dependent and influenced by the time interval between ASO administration and inflammatory stimulation. Subsequent experiments tested whether ASO suppression of SAA was sufficient to mitigate amyloid deposition. Amyloidosis was induced by amyloid-enhancing factor and silver nitrate injection; ASO treatment was initiated 1 week later and continued 1× or 3× per week; inflammation was re-triggered by subsequent injection(s) of silver nitrate; mice were sacrificed after 4–5 weeks. Examination of tissues by Congo red staining and SAA/AA immunohistochemistry revealed consistently less amyloid in the organs of ASO-treated mice compared to saline-treated counterparts. These findings provide rationale for further investigation of SAA-specific ASOs as a potential therapy for AA amyloidosis.

Pathogenesis of experimental amyloid protein A amyloidosis in sore hocks-affected rabbits

Although the experimental transmission of amyloid protein A (AA) amyloidosis with amyloid-enhancing factor has been studied intensively, its pathogenesis remains obscure. We previously found that rabbits affected with ‘sore hocks’ (SH) uniquely developed AA amyloidosis in response to primary inflammatory stimulation followed by the administration of bovine AA fibrils. However, it is unknown why only the rabbits with preexisting SH developed experimental AA amyloidosis. There may be hidden factors in the SH status that stimulate the mechanism of cross-species transmission of AA amyloidosis. To examine the essential factors in the development of experimental AA amyloidosis in SH-affected rabbits, we studied the etiology of SH in rabbits pathologically and bacteriologically. In addition, we developed artificial SH symptoms in normal rabbits by use of an adjuvant prepared from Staphylococcus aureus (StA) isolated from a spontaneous SH-affected rabbit, and we evaluated the incidence of AA amyloidosis in rabbits with or without experimental SH symptoms. We found that StA administration was extremely efficient at stimulating the induction of experimental AA amyloidosis, and the influence of SH was required. We found that the persistent S. aureus infection in SH facilitates the development of experimental AA amyloidosis in rabbits and that the inflammatory stimulation provided by SH acts as an additional accelerator in experimental AA amyloidosis.

Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.