K. Obayashi

Long-term survival after liver transplantation in patients with familial amyloid polyneuropathy

Objective: Familial amyloid polyneuropathy (FAP), which is a fatal disorder inherited in an autosomal dominant fashion, is characterized by systemic accumulation of polymerized transthyretin (TTR) in the peripheral nerves and systemic organs. Liver transplantation has become an accepted treatment of this disorder because it stops the major production of amyloidogenic TTR. However, improved survival of transplant patients compared with that of nontransplant patients has not been sufficiently demonstrated. This study investigated whether transplantation improved the long-term outcome of patients by comparing the survival of patients who had transplantations with that of patients who had not had transplantations. Methods: Eighty consecutive patients with FAP Val30Met who visited Kumamoto University Hospital between January 1990 and December 2010 were studied. The transplant group consisted of 37 patients who had a partial hepatic graft via living donor transplantation in Japan or who underwent liver transplantation in Sweden, Australia, or the United States. The nontransplant group consisted of 43 patients with FAP. Survival was evaluated by using Kaplan-Meier analysis, and the difference in survival was examined via the log-rank test. Results: The transplant group had prolonged survival (p < 0.001) compared with the nontransplant group. The estimated probability of survival at 10 years was 56.1% for the nontransplant group vs 100% for the transplant group. Conclusion: Liver transplantation should be considered as an effective treatment in clinical management of patients with FAP Val30Met. Classification of evidence: This study provides Class III evidence that liver transplantation prolongs survival in patients with FAP Val30Met.

A homozygote case of familial amyloid polyneuropathy amyloidgenic transthyretin Val30Met in a non-endemic area.

Homozygotic cases of familial amyloid polyneuropathy (FAP) are expected to show late onset and mild clinical manifestations, although a homozygote case of FAP showing extremely early onset and severe manifestations was reported recently. Clinical aspects of homozygotic cases of FAP remain controversial. We report a clinical feature of a homozygotic case of FAP in a non-endemic area. The case presented late onset, slow progression, an initial symptom of visual loss, small fiber neuropathy, and autonomic dysfunction. Serum total transthyretin (TTR) levels were relatively higher than those of heterozygotic cases of FAP. Homozygotic combination of stable tetramer of ATTR may have contributed to late onset and slow progression in this case. Introduction: Familial amyloid polyneuropathy (FAP), a fatal inherited autosomal dominant disorder, is characterized by systemic accumulation of polymerized amyloidogenic transthyretin (ATTR) in the peripheral nerves and systemic organs [1,2]. It has recently been suggested that instability of heterozygotic tetramer of variant transthyretin (TTR) structures causes dissociation of the tetramer and amyloid fibril formation [3–5]. It was reported that homozygotic tetramer of TTR was more stable than heterozygotic. Homozygotic cases of FAP are expected to show late onset and mild clinical manifestations [6– 8]. However, a homozygote case of FAP showing extremely early onset and severe manifestations was reported recently [9]. Clinical aspects of homozygotic cases of FAP remain controversial. We report clinical features of a homozygotic case of familial FAP ATTR Val30Met in a non-endemic area. Case report: A 72-year old Japanese female living in Hiroshima was admitted to Kumamoto University Hospital in June 2009 suffering from bilateral vitreous opacities and polyneuropathy. She suffered from paroxysmal atrial fibrillation at the age of 62 and cystitis at the age of 67. In her familial history, her aunt had complete AV block at the age of 70 and dysesthesia in her lower extremities at the age of 85. Her parents are cousins and did not have any relations in an endemic area of FAP foci. The patient had slow progressive visual loss at the age of 58. She developed vitreous opacities at the age of 59 but dysesthesia only at the age of 68. Upon admission, physical examination revealed normal modified body mass index (mBMI) (836 kg g/l m), mild orthostatic hypotension, and edema in left lower leg. Neurological examinations revealed visual loss, pupil border irregularity, dry eye, distal dominant muscle atrophy, and weakness in her lower extremities, absent Achilles’ tendon reflexes, dysesthesia and decreased pain and light touch sensation under knees and elbow with dissociated sensory loss, alternating constipation and diarrhea, and stress urinary incontinence. Total serum TTR were 21.8 mg/dl; relatively higher than those usually found in FAP ATTR Val30Met patients. The case did not present hypothyroidism. Nerve conduction studies showed normal motor nerve conduction velocity, decreased compound muscle action potential (CMAP), and absent sensory nerve action potential (SNAP) in the left ulnar nerve. CMAPs and SNAPs in the lower limbs were not observed. Holter ECG showed PACs and PVCs. R-R interval analysis revealed decreased CVRR (1.44, normal values42.81). Echocardiography did not show either granular sparkling sign, thickening of the intra ventricular septum, nor dilatation of left atrial diameter. Abdominal echography did not show reverse liver–kidney contrast indicating no renal involvement [1–3]. I-MIBG myocardial scintigraphy showed normal accumulation in the heart (early H/M ratio: 3.38, delayed H/M ratio1⁄4 3.13, washout rate 37.3%). Laser Doppler skin blood flow showed vascular hyper reactivity after deep breathing in especially lower extremities. On ophthalmological examination, visual acuities were decreased (0.05 and 0.02) and ocular tensions were normal (5 mmHg and 16 mmHg). Schirmer’s test (3 mm) revealed decreased tear production. Irregularity and amyloid deposition were observed on the pupillay margins. Funduscopic analysis revealed vitreous opacities caused by amyloid deposition. Histopathological examination revealed slight amyloid deposition on the gastric mucosa with Congo red staining under polarized light, although amyloid deposition was not detected in the duodenum mucosa or abdominal fat. After confirming the immunoreactivity of TTR in the amyloid deposits of gastric mucosa using an anti-TTR polyclonal antibody, surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI TOFMS) was used. A single peak corresponding to ATTR Val30Met was detected in the serum. Direct sequence analysis confirmed the homozygotic mutation in the TTR gene presenting one base substitution at codon 30 from GTG (Val) to ATG (Met). Discussion: We report a clinical feature of a homozygotic case of FAP in a non-endemic area. This case showed late onset, slow progression, an initial symptom of visual loss, small fiber neuropathy, 169

Near-infrared spectrophotoscopy of finger venules in assessment of autonomic dysfunction

The authors evaluated morphologic changes in the venules of the finger using near-infrared spectrophotoscopy in patients with autonomic dysfunction, such as familial amyloidotic polyneuropathy and multiple-system atrophy. Abnormalities of the venules, such as tortuosity, irregular venous caliber, and microaneurysm-like change, and a linear negative correlation between the degree of orthostatic hypotension and the degree of vasoconstriction of the venules were observed.

Effect of liver transplantation on the survival of patients with ordinary onset familial amyloid polyneuropathy in Japan.

This study investigated whether transplantation improves the long-term outcome of transplanted Japanese patients with familial amyloid polyneuropathy (FAP) by comparing their long-term survival to that of non-transplanted patients treated during same time period. Fifty-eight consecutive Japanese FAP Val30Met patients who were examined at Kumamoto University Hospital between January 1990 and December 2008 were studied. Survival from onset of disease was analyzed by Kaplan–Maier analysis and difference in the survivals between the two groups was examined by the logrank test. Survival was significantly prolonged in the transplanted group (p5 0.001), compared with that in the non-transplanted group. The estimated probabilities of survival at 10 years were 45.2% in the non-transplanted group, while that in the transplanted group was 100%. In conclusion, our study found that liver transplantation significantly prolongs survival of FAP Val30Met patients compared to that of non-transplanted FAPVal30Met patients. Introduction: Familial amyloid polyneuropathy (FAP), a fatal inherited autosomal dominant disorder, is characterized by systemic accumulation of polymerized amyloidogenic transthyretin (ATTR) in the peripheral nerves and systemic organs [1,2]. Liver transplantation has been considered an acceptable treatment for this systemic amyloid disorder [3–6]. Liver transplantation removes the major source of variant transthyretin (TTR) production in blood and replaces it with a source of normal TTR [7,8]. However, even after liver transplantation, amyloidosis including cardiomyopathy can progress in some patients, perhaps due to deposition of amyloid fibrils derived from wild-type TTR in blood [9,10]. To date, improvement in survival from disease onset in transplant patients compared to that of non-transplanted subjects has not been well demonstrated. The purpose of this study was to investigate whether transplantation improves the long-term outcome by comparing the long-term survival of transplanted Japanese FAP patients with that of non-transplanted patients treated during the same period. Patients and methods: Fifty-eight consecutive Japanese FAP Val30Met patients who were examined at Kumamoto University Hospital between January 1990 and December 2008 were studied. The transplanted group consisted of 32 patients (age at onset 33.9+ 8.0 years, duration before transplantation 2.5+ 6.0 years, male 43.8%) who could find a donor in their family for living donor liver transplantation with partial hepatic graft or who could afford to pay for liver transplantation in Sweden, Australia, or the USA. Transplantations were performed between 1994 and 2008. The non-transplanted group consisted of 26 patients (age at onset 47.0+ 15.7 years, male 46.2%). The primary end point was patient death. Survival from the onset of disease was analyzed by Kaplan–Maier analysis and difference in the survivals between the two groups was tested by the log-rank test. Results: Survival was significantly prolonged in the transplanted group (p5 0.001), compared with that in the non-transplanted group. The estimated probabilities of survival at 10 years were 45.2% in the non-transplanted group, while that in the transplanted group was 100%. Discussion: To our knowledge, this is the first report to compare the survival of ordinary onset FAP Val30Met patients between a transplanted group and a non-transplanted group treated during the same period. Our study demonstrated that liver transplantation significantly prolongs the survival of FAP Val30Met patients, leading to the speculation that liver transplantation inhibits the progression of clinical manifestations of FAP and that liver transplantation might also be effective for FAP patients with other types of ATTR mutations. To clarify the exact effect of therapies, selection bias should be eliminated as strictly as possible. The selection bias due to choosing patients with a good prognosis was slight in this study. Because of a lack of donors in Japan, patients who could pay the cost underwent cadaveric liver transplantation abroad while patients with an available donor underwent liver transplantation in Japan. Patients who could neither pay for a transplant abroad nor provide a donor were not transplanted. A further randomized, double-blind, controlled trial is needed to carefully evaluate the efficacy and safety of liver transplantation for FAP, although such a trial would be problematic for ethical reasons. Conclusion: Our study found that liver transplantation prolongs the survival period of FAP Val30Met patients. Longer studies are needed to assess the 185

Novel homozygous mutation of the caveolin-3 gene in rippling muscle disease with extraocular muscle paresis

We describe a 39-year-old Japanese man with rippling muscle disease who carried a novel homozygous mutation (Trp70 to a stop codon) in the caveolin-3 gene. The patient also had extraocular muscle paresis showing atrophy of the extraocular muscles on orbital MRI. The involvement of the extraocular muscles of patients with caveolinopathy is discussed.

Transthyretin-derived amyloid deposition in the heart of an elderly Japanese population.

Although in Western countries the prevalence of senile systemic amyloidosis (SSA) in the elderly (480 years) was found to be about 25% based on examination of autopsy-derived cardiac specimens, prevalence in Asian countries is still unclear. The aim of the study was to identify clinicopathological features in Japanese patients with transthyretin (TTR)-derived amyloid deposition. Autopsy-derived cardiac specimens were examined. Pathological analyses revealed that TTR-derived amyloid deposition was found in 11% of the patients over 80 years of age. Although the prevalence of TTR-derived amyloid deposition was lower in a Japanese population than that in a Western population, we must consider SSA in elderly patients if the clinical manifestations cannot be explained by other pathologies. Introduction: Amyloidosis is a clinical disorder caused by extracellular deposition of insoluble abnormal fibrils, derived from normally soluble proteins. So far, 27 different precursor proteins have been identified in different kinds of amyloidosis [1]. Transthyretin (TTR) is one of the amyloidogenic proteins and causes two types of amyloid diseases. One is familial amyloid polyneuropathy (FAP), which is hereditary amyloidosis caused by mutatedTTR [2]. The other is non-hereditary senile systemic amyloidosis (SSA) in which wild-type (WT) TTR forms amyloid deposits especially in cardiac and pulmonary tissues, and occasionally in other systemic organs in the elderly [3]. Several reports showed that the prevalence of SSA was up to 25% in the elderly over the age of 80, based on examination of autopsy-derived cardiac specimens in the United State and Europe [4–6]. It has been well documented that most patients with SSA show a slowly progressive amyloid cardiomyopathy which causes congestive heart failure and arrhythmia. However, clinicopathological features of SSA in Asian countries have still not been clearly identified. In this report, we examined the frequency of TTRderived amyloid deposition using autopsy specimens of patients who had not been diagnosed with amyloidosis before their death and the incidence was compared to aging. In addition, the relationship between amyloid deposition and clinical manifestations was also discussed. Materials and methods: Materials: We examined autopsy-derived cardiac specimens of 175 consecutive individuals over 40 years of age at Kumamoto University Hospital, Japan between January 2001 and March 2008. FAP patients were excluded by genetic testing and/or clinical findings in medical records. Concerning age, 10 cases were in their forties, 33 in their fifties, 43 in their sixties, 63 in their seventies, 20 in their eighties, and 5 in their nineties; one case was over 100 years old. In cases with TTR amyloid deposits in cardiac specimens, other tissues were also examined. For immunohistochemical staining, an antibody for human TTR was purchased from Dako (Glostrup, Denmark). Other chemicals used in the study were purchased from Nakarai tesque (Kyoto, Japan). Congo red staining: Tissue samples were fixed with 10% formalin, embedded in paraffin, serially sectioned at a thickness of 4 mm, and placed onto microscopic slides. Sections were stained with alkaline Congo red and hematoxylin. Amyloid deposits were confirmed under polarized light for the presence of green birefringence. Immunohistochemical stainings: The specimens with amyloid deposits were used for immunohistochemical staining. The specimens were deparaffinated, dehydrated in a modified alcohol series, and incubated in blocking buffer (1% bovine serum albumin (BSA), and 5% goat serum in PBS). A polyclonal rabbit anti-human TTR antibody diluted 1:100 in blocking buffer was used as the primary antibody. A horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:100 in blocking buffer was used as the secondary antibody. Reactivity was visualized with the DAB Liquid System (DAKO), according to the manufacturer’s instructions. Sections were counterstained with hematoxylin. For parallel control sections, primary antibody was replaced by blocking buffer. Other immunohistochemical stainings with antibodies for other amyloid precursor proteins were not examined. Results: Congo red staining for the specimen revealed Congo red positive deposition in 17 of 175 cases (9.7%). In the 17 amyloid positive cases, we found TTR-derived amyloid deposition in 5 (2.9%). Immunohistochemical staining with antiTTR antibody was negative in another 12 cases. The incidence of cardiac amyloidosis increased with aging. The incidence of TTR-derived amyloid deposition increased with age: 2 of 63 cases in their seventies, one of 20 cases in their eighties, one of 5 cases in their nineties, and one of one case aged over 100 (Figure 1). In 175 autopsy cases, TTR-derived amyloid deposition was not detected under the age of 69 years. 180

Age-dependent increase in thiol conjugated forms of transthyretin (TTR) in the elderly: quantitative analyses by the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) protein chip system.

Senile systemic amyloidosis (SSA) caused by wild-type (WT) transthyretin (TTR) is a prevalent disorder in the elderly. However, mechanisms of WT TTR amyloid formation and risk factors for SSA remain to be elucidated. In this study, to determine age-related changes in serum TTR, we investigated TTR concentrations and modifications ratios in sera of the elderly. Neither significant age-related changes nor gender-related distinctions in serum TTR concentrations were observed from the age of 60’s to 90’s. In addition, serum TTR concentrations of SSA patients did not significantly change compared to those in coeval elderly. However, all kinds of thiol conjugated-TTR ratios significantly increased as the age by means of surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry, while those of SSA patients did not significantly differ from those of the coeval elderly. We speculate that those thiol conjugations of TTR are one of the required trigger for SSA in the elderly. Introduction: Senile systemic amyloidosis (SSA) is a sporadic transthyretin (TTR) amyloidosis derived from wild-type TTR [1]. It has been well documented that most of the patients with SSA show slowly progressive amyloid cardiomyopathy, which causes congestive heart failure and arrhythmia [1]. Several reports exhibited that the prevalence of SSA was up to 25% in the elderly older than 80 years of age based on examination of autopsy-derived cardiac specimens in the United States and Europe [2]. However, the pathogenesis of SSA remains to be elucidated. TTR is mainly produced in the liver and is present in plasma normally as a homotetramer. In analysis of serum samples, several conjugated forms of TTR were observed in addition to unconjugated forms of TTR [3]. Cys-10 residue in TTR is generally one of the modification sites, and the cys-conjugated form of TTR is found most frequently in serum [4,5]. In addition, other modifications of TTR, such as Ssulfonation, cysteinylglycylation, and glutathionylation, are also detected. It was reported in in vitro study that those modifications increased amyloidogenicity of TTR. However, age-dependent changes of such TTR modifications in the elderly and SSA patients were not still determined. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) combines chromatography with mass spectrometry and uses arrays with different surface chemistries. Chromatographic surfaces of ProteinChips provide good support for co-crystallization of matrix and target proteins, which results in formation of a homogeneous layer on the spot. SELDI-TOF MS instrument is also especially adapted for quantitative assay of TTR molecule [6]. In this study, to determine age-related changes in serum TTR, we investigated TTR concentrations and thiol conjugations of TTR in sera of the elderly by means of nephelometry and SELDI-TOF MS, respectively. Materials and methods: Serum samples and measurement of serum TTR concentrations: For measuring TTR concentrations, 409 serum samples of elderly patients (60’s: 136, 70’s: 140, 80’s: 114, and 90’s: 19) and 6 serum samples of SSA patients (74.5+ 5.5 years old) were employed. We excluded patients with low albumin concentrations (54.1 g/dl) and/or high C-reactive protein concentrations (40.3 mg/dl) in sera from the study. Serum TTR concentrations were measured by nephelometry. For evaluation of TTR modifications, we used each 10 serum samples of patients in 60’s, 70’s, 80’s and 90’s, respectively. Informed consent was obtained from each subject. All studies using serum samples were in accordance with the current revision of the Helsinki Declaration. Figure 1. Serum TTR concentrations in the elderly and SSA patients. Serum samples of 409 elderly patients and 6 SSA patients (74.5+ 5.5 years) were employed. Serum TTR concentrations were measured by nephelometry. 14

Relationship between amyloid fibril formation and overproduction of basement membrane components in familial amyloidotic polyneuropathy (FAP).

In transthyretin (TTR)-related amyloidosis, microenvironmental factors have been thought to play an important role in amyloidogenesis; however, the details of these interactions remain largely unknown. We analyzed the relationship between amyloid fibril formation and microenvironmental changes especially in the basement membrane, by using autopsy cardiac tissues from familial amyloidotic polyneuropathy (FAP) patient and a cultured cell line. Histopathological analyses revealed that TTR amyloid fibrils first formed and grew at the basement membrane of cardiomyocytes. Cell culture experiments and measurement of a serum marker for basement membrane suggested that amyloid fibrils induced up-regulation of the biosynthesis of basement membrane components, which contributed to further amyloid formation. The chain reaction of amyloid fibril formation with induction of basement membrane components may play important roles in FAP amyloidogenesis. Introduction: Familial amyloidotic polyneuropathy (FAP) is caused by a point mutation in the transthyretin (TTR) gene. In vitro studies of TTR amyloidogenesis led to the hypothesis that soluble TTR self-assembles into amyloid fibrils as a result of point mutations or deletions in the TTR gene that thereby lead to amyloidogenic TTR (ATTR). However, other factors such as aging, and genetic factors, and environmental factors are thought to be involved in the pathogenesis of FAP. Many pathological studies of FAP, especially FAP ATTR Val30Met, revealed that TTR amyloid demonstrated organ and tissue tropism [1]. The in vivo TTR amyloid fibril formation mechanism seems to be closely related to the microenvironment where amyloid deposits occur. In the heart, which is one of the most common organs of amyloid deposition in FAP as well as senile systemic amyloidosis, amyloid fibrils predominantly deposit at the basement membrane surrounding each cardiomyocyte. High-affinity interactions between amyloid precursor proteins and basement membrane components were previously reported, and basement membrane is thought to play an important role as a scaffold for amyloid fibril formation process [2]. To elucidate the pathogenesis of TTR amyloid fibril formation in vivo, we analyzed the relationship between amyloid fibril formation and microenvironmental changes, by using autopsy cardiac tissues from FAP patient and a cultured cell line. Methods: Light microscopy: We examined autopsy cardiac tissues from 11 patients with FAP ATTR Val30Met in histopathological analyses. Sections were stained with Congo red Immunohistochemical analyses were performed using rabbit polyclonal anti-TTR, mouse monoclonal anti-collagen IV, rabbit polyclonal anti-laminin, and rabbit polyclonal anti-fibronectin primary antibodies. The secondary antibody was a horseradish peroxidaseconjugated goat anti-rabbit or rabbit anti-mouse immunoglobulin antibody. Reactivity was visualized with the DAB Liquid System. Electron microscopy: Ultrathin sections were stained with 4% uranyl acetate and lead citrate and were examined with a transmission electron microscope. Cell culture experiment: TTR amyloid fibrils were generated from wild-type-TTR (WT-TTR) and Val30Met TTR under acidic condition. The human vascular smooth muscle cell line glomotel was cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum. Freshly prepared 20 mM native TTR or TTR fibrils were added, and the plates were incubated for an additional 48 h. Cells were rinsed with PBS, and total RNA was isolated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed with the LightCycler 480 system using primers for collagen IV, laminin, and GAPDH. Measurement of serum collagen IV levels: Serum samples were obtained from patients with FAP Val30Met (n1⁄4 9), asymptomatic carriers of the mutation (n1⁄4 7), and healthy volunteers (n1⁄4 9). Serum levels of collagen IV were measured. Results and discussions: We focused on cardiac tissues in this study, because amyloid fibrils predominantly deposit at the basement membrane surrounding each cardiomyocyte. In histopathological analyses, we found small focal amyloid deposits, which represent the Figure 1. Effects of TTR amyloid on mRNA expression of collagen IV and laminin in glomotel cells. RT-PCR analysis indicated expression of collagen IV and laminin mRNAs, normalized to GAPDH as a reference were induced by TTR amyloid. 22

Transthyretin-derived amyloidosis in musculoskeletal systems.

Transthyretin-derived amyloid deposition is commonly found in tenosynovium of senile systemic amyloidosis patients. However, that in ligaments of other organ sites remains to be elucidated. The purpose of this study was to determine the frequency and types of amyloid deposits found in musculoskeletal systems. We investigated patients with carpal tunnel syndrome, rotator cuff tears, and lumbar canal stenosis. We identified 39 cases with TTR amyloid deposits. The mean age in TTR positive cases was higher than non-TTR cases in tenosynovium and yellow ligaments, but not significantly in rotator cuff samples. In the elderly, TTR-derived amyloid deposits were frequently found in ligaments and tendons of various organ sites. Those amyloid deposits may be involved in the pathogenesis of the orthopedic disorders. Abbreviations: FAP1⁄4 familial amyloidotic polyneuropathy; SSA1⁄4 senile systemic amyloidosis; TTR1⁄4 transthyretin Introduction: Amyloidosis is a clinical disorder caused by extracellular deposition of insoluble abnormal fibrils, derived from aggregation of misfolded normally soluble protein [1]. So far, 27 different precursor proteins have been identified in different kinds of amyloidosis [1,2]. It has been well documented that mutated forms of amyloidogenic proteins are more prone to form amyloid fibrils [3]. Transthyretin (TTR) is one of amyloidogenic proteins causing two types of amyloid diseases because TTR itself has b-sheet rich structure and is highly amyloidogenic [1,4]. One is familial amyloid polyneuropathy (FAP), which is hereditary amyloidosis mainly derived from mutated-TTR [5] and the other is senile systemic amyloidosis (SSA). Recently, SSA has been focused in the recent attention [6]. SSA, non-hereditary form of amyloidosis in which wild-type (WT) TTR generates amyloid deposits especially in cardiac and plumonary tissues and occasionally in other systemic organs in the elderly [7]. It has been well documented that most of patients with SSA show a slowly progressive, infiltrative amyloid cardiomyopathy [7]. This disease is believed to be associated with the aging process. In addition, localized type of TTR amyloid has been sometimes reported [8]. However, it was not well known whether it became the pathogenesis of the disease. Accompanied with orthopedic disorders, amyloid deposition in the ligament and tendon has been reported [9], the relationship with disease and the pathogenesis remains to be elucidated. In this report, we examined the frequency of amyloid deposition in tissues resected by operations because of orthopedic disorders. In addition, relationship between amyloid deposition and clinical manifestations were also discussed. Patients and methods: Patients: We investigated 111 specimens of patients with carpal tunnel syndrome (54 specimens), rotator cuff tears (21 specimens), and lumbar canal stenosis (36 specimens). Those patients were diagnosed at the Department of Orthopaedic Surgery in Kumamoto University Hospital and its associated faculties based on clinical finding and radiological examinations from 2008 to 2009. Congo red staining and immunohistochemistry: Formalin-fixed, paraffin-embedded specimens were stained with hematoxylin–eosin and Congo red, as described previously [3]. Elucidation of FAP: To exclude TTR-related FAP, we performed genetic testing, such as a real time PCR or sequencing. Mass spectrometry using surface-enhanced laser desorption/ionization time-offlight mass spectrometry (SELDI/TOF MS) [10] was performed to confirm the patients who did not posses variant TTR in serum. Ethics: The study protocol was approved by Human Ethics Review Committee of Kumamoto University and a signed consent form was obtained from the family of subjects. All patients’ family members gave their informed consent for performing an autopsy. Results: The presence of amyloid deposits was determined by a positive reaction in paraffin sections with Congo red staining, and by an apple green birefringence in the same sections in polarized light. Histochemical analysis with Congo red staining revealed 47 (39%) amyloid positive samples (Figure 1A). Immunohistochemical examination was performed using anti-amyloid precursor proteins as mentioned in section ‘Materials and method’. AntiTTR antibody reacted with the lesions where amyloid deposition was positive in 39 cases (Figure 1B): 18 cases in flexor tenosynovium, 5 cases in rotator cuff tendons, and 16 cases in the yellow ligaments. In eight samples, any antibodies 163

Antioxidative effect of albumin on amyloid fibril formation in transthyretin-related amyloidosis.

Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP) is characterized by systemic accumulation of amyloid fibrils caused by a point mutation in the TTR gene. Despite the urgent need for alternative therapeutic strategies, the pathogenesis of FAP still remains unclear. In this study, we focused on albumin, the most abundant protein in plasma, and reported the role of albumin in the TTR amyloid formation process. Serum albumin levels were significantly decreased in FAP patients as the disease progressed. N-ethylmaleimide-albumin, diminishing the antioxidant effect, abolished its inhibitory effect on amyloid formation, suggesting that albumin as an antioxidant effectively suppressed TTR amyloid formation. In FAP patients, albumin was significantly oxidized as the disease progressed. Moreover, loss of functional albumin accelerated TTR deposition in analbuminemia rats with a human variant TTR gene. Taken together, these results indicate that albumin, as an antioxidant, may play an inhibitory role in the process of TTR amyloid formation. Introduction: Familial amyloidotic polyneuropathy (FAP), which is induced by amyloidogenic transthyretin (ATTR), is characterized by systemic accumulation of amyloid fibrils in the peripheral nerves and other organs [1]. Although many studies have been carried out to identify various types of ATTR-related FAP, the precise mechanism of TTR amyloid formation remains to be clarified. TTR normally behaves as a soluble tetramer and binds to retinol binding protein and thyroxine (T4) in plasma. It has been proposed that tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a compact non-native monomer with low conformational stability can lead to amyloid fibril formation. In addition, post-translational modification of TTR is also recognized as playing a key role in amyloid fibril formation. The involvement of oxidative stress in amyloid fibril formation in tissues of FAP patients has been reported [2]. Our previous in vitro study showed that nitric oxide-mediated modification of TTR may play an important role in amyloid formation, indicating that oxidative stress facilitates amyloid formation [3]. Albumin is the most abundant protein in plasma. It serves as a transport carrier for various ligands and represents the major and predominant antioxidant in plasma [4]. Previous studies have shown that total reactive antioxidant potential in plasma, considered as an index of the level of antioxidants, was decreased in patients with FAP [5]. In addition, more recent studies have demonstrated that albumin suppressed amyloid formation of amyloid-b (Ab), a component of amyloid fibril in Alzheimer’s disease, through decreasing oxidative stress [6]. This evidence suggests that albumin as an antioxidant may play a crucial role in amyloid formation in FAP. In this study, we clarified the role of the antioxidative property of albumin in FAP disease progression. Methods: Both wild type (WT)-TTR and ATTRV30M were purified from serum samples obtained from healthy volunteers and homozygotic FAP ATTR V30M patients. Albumin was isolated from serum samples from healthy volunteers by ionexchange chromatography. Function of albumin isolated from FAP ATTR V30M patients was analyzed quantitatively and qualitatively. To evaluate the effect of albumin on amyloid formation, sandwich ELISA was performed by using monoclonal anti-TTR115-124 antibody, which specifically reacts with amyloid fibrils and preamyloid deposits [7]. Nethylmaleimide (NEM)-albumin was prepared by treatment with charcoal and NEM. To evaluate the effect of albumin on TTR deposition in vivo, analbuminemic transgenic rats possessing a human ATTR V30M gene (V30M Tg NAR) were analyzed. Results and discussion: The serum albumin levels were significantly decreased in FAP patients as the disease progressed. The effect of albumin on amyloid formation was assessed by sandwich ELISA. Of various serum proteins, albumin showed potent inhibition of amyloid formation, compared with alpha1-acid glycoprotein and transferrin. The amyloid formation of both WT-TTR and ATTR-V30M was suppressed in the presence of albumin in a dosedependent manner. These data suggest that albumin may be closely associated with the process of TTR amyloid formation. To clarify the antioxidant effect of albumin on TTR amyloid formation, we next assessed the effect of modified albumin, which was modified in its antioxidant effect. NEM-albumin, diminishing the antioxidant effect, abolished its inhibitory effect on amyloid formation, suggesting that the antioxidative 17