Systemic Amyloid Research Articles

Life Science Applications Utilizing Radiocarbon Tracing

Radiocarbon-based accelerator mass spectrometry (AMS) facilities at Uppsala University include a measurement center for archaeological applications and a separate entity dedicated to life science research. This paper addresses the latter, with the intention of giving a brief description of the biomedical activities at our laboratory, as well as presenting new data. The ultra-small sample preparation method, which can be used down to a few μg C samples, is outlined and complemented with new results. Furthermore, it is shown that the average secondary ion current performance for small samples can be improved by increasing the distance between the cathode surface and the pressed graphite surface. Finally, data is presented for a new application: Amyloidoses are a group of diseases where the conformational changes in specific proteins' structure lead to the formation of extracellular deposits that spread and increase in mass and eventually may lead to total organ failure and death. The formation timeframe is unknown and yet it is an important clue for the elucidation of the mechanism. We present results on bomb-peak dating of 4 different types of purified amyloid proteins from human postmortem heart and spleen samples. The data indicates that the average measured age of the carbon originating from the systemic amyloid types studied here correspond to a few years before the death of the subject. This suggests that a major part of the fibril formation takes place during the last few years before death, rather than as an accumulation of amyloid deposits over decades.

Life Science Applications Utilizing Radiocarbon Tracing

Radiocarbon-based accelerator mass spectrometry (AMS) facilities at Uppsala University include a measurement center for archaeological applications and a separate entity dedicated to life science research. This paper addresses the latter, with the intention of giving a brief description of the biomedical activities at our laboratory, as well as presenting new data. The ultra-small sample preparation method, which can be used down to a few μg C samples, is outlined and complemented with new results. Furthermore, it is shown that the average secondary ion current performance for small samples can be improved by increasing the distance between the cathode surface and the pressed graphite surface. Finally, data is presented for a new application: Amyloidoses are a group of diseases where the conformational changes in specific proteins' structure lead to the formation of extracellular deposits that spread and increase in mass and eventually may lead to total organ failure and death. The formation timeframe is unknown and yet it is an important clue for the elucidation of the mechanism. We present results on bomb-peak dating of 4 different types of purified amyloid proteins from human postmortem heart and spleen samples. The data indicates that the average measured age of the carbon originating from the systemic amyloid types studied here correspond to a few years before the death of the subject. This suggests that a major part of the fibril formation takes place during the last few years before death, rather than as an accumulation of amyloid deposits over decades.

Amyloidosis cutis dyschromica

BackgroundAmyloidosis cutis dyschromica is a rarely documented variant of cutaneous amyloidosis. To date, only 26 cases have been reported.ObjectiveThe purpose of this study was to improve the clinical and histopathological data for this variant of amyloidosis and to highlight the immunohistochemical features of the disease. The published cases were also reviewed.MethodsWe performed a retrospective review of patients with amyloidosis cutis dyschromica in a single centre. The clinical, histopathological and immunohistochemical features were documented and analysed.ObservationsWe described 10 cases of amyloidosis cutis dyschromica. Six of them were female. Five patients were from the same family, and the other 5 were sporadic. The distinguishing features of the clinical presentation included generalised mottled hyper- and hypopigmented macules, which were asymptomatic or mild pruritic. The typical onset of the lesions occurred in childhood (n = 7) and occasionally after puberty (n = 3). No evidence of systemic amyloidosis deposition was observed in these cases of amyloidosis cutis dyschromica. Amyloid deposits were observed in the papillary dermis and were positive for the Congo red stain. An immunohistochemical study showed that the amyloid expresses cytokeratins CK34βE12 and CK5/6.ConclusionsWe described the largest series of amyloidosis cutis dyschromica to date and reviewed the published patients. This rare disease is featured by generalised mottled hyper- and hypopigmented lesions, and it is a rare variant of primary cutaneous amyloidosis without evidence of systemic amyloid deposition. Positive staining for the cytokeratins CK34βE12 and CK5/6 in amyloidosis cutis dyschromica suggests that the amyloid is derived from keratinocytes.

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N-terminal 76-amino acid residue-long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases--human cystatin C (hCC)--has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA-hCC complex, using a combination of selective proteolytic excision and high-resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86-104), whereas the binding site in hCC sequence was identified as hCC(96-102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA-hCC complex.

A new lysozyme tyr54asn mutation causing amyloidosis in a family of Swedish ancestry with gastrointestinal symptoms

Familial amyloidoses are a group of inherited disorders that cause deposition of misfolded amyloidogenic proteins in various tissues, resulting in organ dysfunction. Point mutations in the coding region of seven different genes are known to cause clinically significant systemic amyloid disease. We describe a new mutation in exon 2 of the lysozyme gene associated with amyloidosis (ALys) in a 61-year-old woman with a 7-year history of non-bloody, watery diarrhea, and weight loss. Biopsies of the duodenum and stomach were positive for amyloid deposits in the lamina propria and blood vessels. Direct DNA sequencing of the lysozyme gene revealed a single base nucleotide transversion from T to A at the first position of codon 54, resulting in replacement of Tyr by Asn in the mature lysozyme protein (pTyr54Asn). Immunoblot analysis of amyloid fibrils extracted from a fat tissue sample confirmed lysozyme as the amyloid protein. Clinically, the phenotype associated with this lysozyme mutation featured chronic abdominal pain, diarrhea, weight loss, malabsorption, and sicca syndrome. There was no associated nephropathy as has been reported for other ALys mutations. We describe a new mutant lysozyme that presents with abdominal discomfort, diarrhea, weight loss, and sicca syndrome.

Familial amyloidotic polineuropathy and systemic lupus

Familial amyloidotic polineuropathy is a genetic disorder, leading to systemic amyloid deposits, manifested as sensory-motor and autonomic neuropathy. In the Portuguese classical form, the disease is evident at a young age, and causes death if no specific treatment is received. Variability in penetrance, age of onset and clinical course has been published; environmental and genetic factors are believed to contribute to this variability. The authors report a case of a 51-year-old white female, with a medical history of acquired angioedema, late-onset familial amyloidotic polineuropathy and systemic lupus erythemathosus. The authors consider that these associated diseases could modulate their expression.

The repertoire of λ light chains causing predominant amyloid heart involvement and identification of a preferentially involved germline gene, IGLV1-44

AbstractMonoclonal Ig light chains (LC) can be responsible for pathologic conditions in humans, as in systemic amyloid light amyloidosis. Protean clinical manifestations characterize this disorder with the most varied combination of symptoms generated by different degrees of diverse organ involvement. Kidney and heart are most frequently interested, with major heart involvement as the most relevant prognostic factor. The identification of the underlying mechanism involved in organ targeting is of major relevance for the pathobiology of this disorder. To this aim, we characterized the repertoire of variable region germline genes of λ LC preferentially targeting the heart and compared it with the repertoire of LC that do not in a case-control study. We found that the repertoires were highly restricted, showing preferential use of the same few germline genes but with a different frequency pattern. A single gene, IGVL1-44, was found associated with a 5-fold increase in the odds of dominant heart involvement (after adjusting for confounders in a multivariable logistic model). These results support an involvement of LC genetics in the determination of organ targeting. Study of the characteristics of IGVL1-44-LC with, and of the minority without, heart involvement might lead to identification of LC/tissue interactions.

Hidradenitis suppurativa resulting in systemic amyloid A amyloidosis: A case report and review of the literature

Hidradenitis suppurativa is a chronic, inflammatory disease of the follicular epithelium that presents as tender, subcutaneous nodules in an intertriginous distribution with sinus tract formation. Most commonly, hidradenitis suppurativa results in local complications, such as scarring and infection. However, systemic complications, such as anemia and arthropathies, have also been described. Herein, we report an unusual case of systemic amyloid A secondary to hidradenitis suppurativa. We describe a 39-year-old man with a long history of recurrent, tender, erythematous nodules in the axillary and anogenital regions, resulting in abscesses, sinus tract formation, and large areas of scarring. After 21 years of cutaneous disease with concurrent elevated systemic inflammatory markers, the patient was noted to have significant proteinuria. A kidney biopsy and immunostaining revealed deposits of amyloid A. Echocardiogram and electrocardiogram showed ventricular and atrial wall thickening with an appearance consistent with cardiac amyloid deposition. Systemic amyloid A amyloidosis is a serious, but rare, complication of chronic inflammatory disorders, including hidradenitis suppurativa, with potential multi-organ involvement including renal and cardiac manifestations. Amyloid A amyloidosis should be suspected in patients with chronic inflammatory cutaneous diseases who present with renal abnormalities, especially proteinuria or the nephrotic syndrome.

Renal amyloidosis caused by apolipoprotein A-II without a genetic mutation in the coding sequence

Although the majority of renal amyloidosis is caused by either acquired monoclonal immunoglobulin light-chain amyloidosis or reactive systemic amyloid A, some cases are caused by hereditary amyloidosis. Apolipoprotein A-II (apoAII) amyloidosis is a rare form of hereditary amyloidosis and cannot be diagnosed by a routine examination. Thus, the prevalence and etiology of apoAII amyloidosis are uncertain. In humans, a genetic mutation in the stop codon of apoAII is considered to be a cause of amyloid fibril formation. We report on a 68-year-old man who presented with proteinuria by apoAII amyloidosis without family history. His proteinuria gradually increased to 6g/day within 1year. A renal biopsy showed amyloid deposition in the glomeruli, however, acquired monoclonal immunoglobulin light-chain amyloidosis and reactive systemic amyloid A were ruled out. Immunohistochemistry revealed apoAII deposition in the glomeruli, but DNA sequencing did not identify any genetic mutation in the coding sequence of apoAII. Here, we report a case of apoAII amyloidosis without a genetic mutation in the coding sequence and discuss the etiology of apoAII amyloidosis.

Neuronal Production of Transthyretin in Human and Murine Alzheimer's Disease: Is It Protective?

Transthyretin (TTR), a systemic amyloid precursor in the human TTR amyloidoses, interacts with β-amyloid (Aβ) in vitro, inhibits Aβ fibril formation, and suppresses the Alzheimer's disease (AD) phenotype in APP23 mice bearing a human APP gene containing the Swedish autosomal dominant AD mutation. In the present study, we show that TTR is a neuronal product upregulated in AD. Immunohistochemical analysis reveals that, in contrast to brains from non-demented age-matched individuals and control mice, the majority of hippocampal neurons from human AD and all those from the APP23 mouse brains contain TTR. Quantitative PCR for TTR mRNA and Western blot analysis show that primary neurons from APP23 mice transcribe TTR mRNA, and the cells synthesize and secrete TTR protein. TTR mRNA abundance is greatly increased in cultured cortical and hippocampal embryonic neurons and cortical lysates from adult APP23 mice. Antibodies specific for TTR and Aβ pulled down TTR/Aβ complexes from cerebral cortical extracts of APP23 mice and some human AD patients but not from control brains. In complementary tissue culture experiments, recombinant human TTR suppressed the cytotoxicity of soluble Aβ aggregates added to mouse neurons and differentiated human SH-SY5Y neuroblastoma cells. The findings that production of Aβ, its precursor, or its related peptides induces neuronal TTR transcription and synthesis and the presence of Aβ/TTR complexes in vivo suggest that increased TTR production coupled with interaction between TTR and Aβ and/or its related peptides may play a role in natural resistance to human AD.

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1⁻/⁻) mice were used. Apoa1⁻/⁻ mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1⁻/⁻ mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1⁻/⁻ mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.

How to treat patients with systemic amyloid light chain amyloidosis? Comparison of high-dose melphalan, low-dose chemotherapy and no chemotherapy in patients with or without cardiac amyloidosis

High-dose melphalan and autologous stem cell transplantation (HDM) is an effective treatment for systemic amyloid light chain (AL) amyloidosis but the eligibility criteria exclude many patients with this disorder. The aim of this study was to determine appropriate treatment strategies for systemic AL amyloidosis according to each patient's clinical condition in Japan. Historical cohort study. Fifty-three patients with systemic AL amyloidosis (those with malignancies were excluded) were treated in our hospital with HDM (15 patients), melphalan + prednisolone (MP) (17 patients), vincristine + adriamycin + dexamethasone (VAD) (11 patients), or supportive treatment (no chemotherapy, 10 patients). We compared the survival rates among these treatment groups. Mean survival was significantly longer in the HDM group than in the other three groups (P<0.01, log-rank test). This trend remained the same when patients were divided into those with and without cardiac amyloid involvement. Furthermore, in patients with heart involvement, survival in the VAD therapy group was significantly inferior to that in the MP therapy group (P<0.01 by log-rank test). Significant factors related to the survival rate included the presence or absence of heart involvement and treatment modality. HDM should be considered the treatment of choice in eligible patients with systemic AL amyloidosis even in the presence of cardiac amyloidosis. If HDM is not eligible, indications for VAD therapy should be carefully evaluated in patients with cardiac amyloidosis.

Current status of hematopoietic cell transplantation in the treatment of systemic amyloid light-chain amyloidosis

Systemic amyloid light-chain (AL) amyloidosis is a protein conformation disorder caused by clonal plasma cell dyscrasias. Symptoms result from fibrillar extracellular deposits in various tissues. The deposits disrupt organ function and ultimately lead to death. The prognosis is poor and depends mostly on the severity of cardiac involvement. The treatment is derived from the therapy of multiple myeloma with the main goal being to reach a complete hematological remission (CR). High-dose melphalan (HDM) and autologous hematopoietic cell transplantation can induce CR rates in about 40%. The main concern was the high transplant-related mortality of up to 40% due to organ dysfunction, which could be reduced to <12% by careful patient selection in experienced centers. However, >50% of patients in CR survive longer than 10 years, suggesting that HDM has the potential to change the natural course of the disease. As there is evidence that 'graft-versus-plasma-cell-dyscrasia' effects are active in AL amyloidosis, allogeneic hematopoietic cell transplantation might be an option for younger patients with preserved organ functions who have relapsed after HDM.

MAJOR SPONTANEOUS RENAL HAEMORRHAGE AS AN UNUSUAL COMPLICATION OF SYSTEMIC AMYLOIDOSIS

We report the case of a patient with systemic amyloidosis who developed a life-threatening spontaneous haemorrhage from an amyloid kidney. A review of the literature suggests that this is a rare complication of renal amyloidosis. However, the grossly abnormal renal angiography observed in our patient has previously been described in the literature. Therefore, spontaneous renal haemorrhage should be considered a risk in any systemic amyloid patient, especially in the differential diagnosis for those patients with the condition who present with an acute abdomen.

Development of amyloidosis in patients with rheumatoid arthritis under TNF-blocking agents

Sir,We have read with great interest the article by Nakamuraet al. [1] describing the beneficial effect of etanercept inpatients with renal amyloidosis secondary to rheumatoidarthritis (RA). Sustained chronic inflammation favors theoccurrence of systemic amyloid A (AA) amyloidosis in RApatients [2] and the control of the inflammatory process isof crucial importance for amyloidosis prevention, as oncethis condition is established, its treatment is often disap-pointing. Some recent reports, including the one byNakamura, have demonstrated a beneficial effect of anti-TNF [3, 4], anti-IL-6 [5], and B cell depletion [6] therapiesin AA amyloidosis. These therapies could thereforerepresent a hope for patients with AA amyloidosissecondary to RA. Unfortunately, biologic agents do notprevent amyloidosis progression or even its development inall patients.We would like to report the onset of AA amyloidosis intwo patients with RA while under TNF-blocking therapy,despite an acceptable inflammatory control.The first case is a 69-year-old male with RA since theage of 64, in whom infliximab was started 1 year after RAdiagnosis, given the inadequate disease control withmethotrexate (20 mg once a week) and prednisolone(5 mg/day). A good EULAR response was achieved andmaintained for the subsequent 3 years, when the investiga-tion of a persistently elevated erythrocyte sedimentationrate (ESR) lead to the diagnosis of secondary amyloidosis33 months after being started on infliximab (Table 1). Thepresence of AAwas histologically confirmed by abdominalfat biopsy. There was no evidence of renal, cardiac, orgastrointestinal involvement. The patient continued on anti-TNF therapy and after 18 months remains free of majororgan involvement.The second patient is a 66-year-old female with RAsince the age of 50. Amyloidosis was diagnosed 14 yearsafter RA onset, while on treatment with etanercept andmethotrexate for 88 months and with a good EULARresponse (Table 1). Also in this case, the suspicion of AAamyloidosis was raised due to a persistently increased ESRdespite the absence of joint or other complaints. Sheunderwent an abdominal fat biopsy that confirmed thepresence of amyloid A.These two cases highlight the possibility of AAamyloidosis development in patients under TNF-blockingagents and with an adequate control of the inflammatorydisease according to the disease activity score (28-jointcount) DAS28. Both patients pursued biological treatmentwithout any relevant adverse events, and without clinicalevidence of AA amyloidosis progression. To date, bothremain free of renal, intestinal, or cardiac impairment. Eventhough TNF-blocking agents did not prevent AA amyloid-osis, their use was safe and might have contributed topatients’ clinical stability and the absence of major organinvolvement.

Spatial Extent of Charge Repulsion Regulates Assembly Pathways for Lysozyme Amyloid Fibrils

Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path.

Pegylated leptin antagonist ameliorates chronic colitis by inducing regulatory T cells (Tregs) partially mediated by TGF-beta induction (162.4)

Abstract IIBD is a chronic intestinal inflammation caused by hyper activated effector immune cells that affects millions of people around the world. Several lines of evidence support an important role for TGF-beta in the induction of tolerance and control gut inflammation. Leptin is satiety hormone produced primarily by adipose tissue, which increases during IBD progression. We investigated the effect of pegylated leptin antagonist mediated inhibition of chronic colitis in IL-10-/- mice. In the present study, we report that pegylated leptin antagonist (mutant L39A/D40A/F41A) effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis including a decrease in body weight, systemic serum amyloid A (SAA), and leptin levels. The percentage of Tregs in the spleen, mesenteric lymph node (MLN) and intestinal lamina propria (LP) of colitis mice was increased after leptin antagonist treatment as compared to control mice. Similarly, the percentage of TGF-beta in the MLN and LP of colitis mice increased after leptin antagonist treatment. Leptin antagonist also reduced systemic and mucosal cytokines as well as increased the insulin level as compared to control mice. These findings suggest that leptin antagonist ameliorates chronic colitis by inducing TGF-beta, which mediates the induction of Tregs and reduces both mucosal and systemic cytokines. The intricate interaction among leptin, TGF-beta and Tregs might provide new strategies for therapeutic interventions.

Generalized Amyloidosis in an Eastern Bongo (Tragelaphus eurycerus isaaci)

Amyloidosis has been described in a wide range of domestic and wild species and man. A 10-yr-old male Eastern (Mountain) Bongo (Tragelaphus eurycerus isaaci) was submitted for postmortem examination after a period of 24-hr malaise. Gross examination found evidence of biventricular cardiac hypertrophy, congestive heart failure, and focal pulmonary abscessation. Histologic changes in the heart were consistent with hypertrophic change. Amyloid deposits were found within the liver, kidney medulla, heart, adrenal cortex, and pituitary gland and were confirmed as reactive systemic amyloid (AA) by immunohistochemistry. The pulmonary abscessation was thought to be the stimulus for excessive serum amyloid associated protein production leading to the reactive systemic amyloidosis. Colloidal goiter was also identified as an incidental finding.

Near Field Detection of Beta-Amyloid Proteins by the use of Olfactory Cells and Nano Particles in a Microfluidic Channel

It is well known that Alzheimer's disease (AD) is pathologically defined by the presence of amyloid-beta plaques and neurofibrillary tangles within the brain by advanced medical imaging technique such as computed tomography or magnetic resonance imaging. Currently, in AD-related olfactory sensory loss studies, early olfactory perceptual loss is likely contributed by nonfibrillar, versus fibrillar, amyloid-beta related mechanism in the olfactory system and nonfibrillar amyloid beta deposition is observed within the olfactory bulb. Therefore, the results of the olfactory dysfunction studies represents that if the amount of nano-sized amyloid beta proteins (nano-Abs) is quantified in in vivo olfactory system, in vivo early diagnosis of AD is possible. In this paper, we have measured the amount of nano-Abs by the use of olfactory cells and nano particles (100nm bead) in a micro-fluidic channel. For sample preparation, nano-particles are sequentially conjugated with linkers (biotin, straptavidin and so on) and the particles are absorbed (or adhere) into in vivo olfactory system. The amount of the conjugated nano-Abs is measured by evanescent field-nano particle coupling effect where we use micro-fluidic channels. As results, in the in vivo olfactory system, the coupled optical power with single nano-particle is around 1nW. The results of quantification with amyloid-related nano-particles in in vivo olfactory system will be briefly introduced.

Tongue necrosis and systemic vascular amyloidosis

Tongue necrosis is a rare but dramatic clinical finding, which is most often related to infection, trauma, malignancy, or vasculitis. We describe an extremely unusual case in which systemic amyloid deposition in small arteries caused by senile systemic amyloidosis was associated with necrosis of the tongue as well as necrosis of portions of the bowel. To our knowledge, this is the first report of ischemic tongue necrosis resulting from vascular amyloidosis.