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ABSTRACT We herein describe a case of severe post‐biopsy bleeding and perforation in gastric amyloidosis. A 70‐year‐old man who had been on dialysis underwent esophagogastroduodenoscopy and biopsy was performed. Post‐biopsy bleeding occurred, and three times of coagulation hemostasis and once clip hemostasis were performed. After the hemostasis, he eventually had a gastric perforation and omental patch surgery was performed, however, he passed away after the surgery. From the pathological finding of biopsy specimen, he was diagnosed with gastric amyloidosis caused by dialysis‐related amyloidosis. In patients of amyloidosis, the risk of bleeding and perforation is elevated due to vascular and tissue fragility. Therefore, when performing hemostasis in patients with gastrointestinal amyloidosis, clip hemostasis which minimizes tissue damage is considered preferable to coagulation hemostasis.

We herein describe a case of severe post‐biopsy bleeding and perforation in gastric amyloidosis. A 70‐year‐old man who had been on dialysis underwent esophagogastroduodenoscopy and biopsy was performed. Post‐biopsy bleeding occurred, and three times of coagulation hemostasis and once clip hemostasis were performed. After the hemostasis, he eventually had a gastric perforation and omental patch surgery was performed, however, he passed away after the surgery. From the pathological finding of biopsy specimen, he was diagnosed with gastric amyloidosis caused by dialysis‐related amyloidosis. In patients of amyloidosis, the risk of bleeding and perforation is elevated due to vascular and tissue fragility. Therefore, when performing hemostasis in patients with gastrointestinal amyloidosis, clip hemostasis which minimizes tissue damage is considered preferable to coagulation hemostasis. [ABSTRACT FROM AUTHOR]

Dialysis-related amyloidosis (DRA) is a severe complication in end-stage kidney disease (ESKD) patients undergoing long-term dialysis treatment, characterized by the deposition of β2-microglobulin-related amyloids (Aβ2M amyloid). To inhibit DRA progression, hexadecyl-immobilized cellulose bead (HICB) columns are employed to adsorb circulating β2-microglobulin (β2M). However, it is possible that the HICB also adsorbs other molecules involved in amyloidogenesis. We enrolled 14 ESKD patients using HICB columns for DRA treatment; proteins were extracted from HICBs following treatment and identified using liquid chromatography–linked mass spectrometry. We measured the removal rate of these proteins and examined the effect of those molecules on Aβ2M amyloid fibril formation in vitro. We identified 200 proteins adsorbed by HICBs. Of these, 21 were also detected in the amyloid deposits in the carpal tunnels of patients with DRA. After passing through the HICB column and hemodialyzer, the serum levels of proteins such as β2M, lysozyme, angiogenin, complement factor D and matrix Gla protein were reduced. These proteins acted in the Aβ2M amyloid fibril formation. HICBs adsorbed diverse proteins in ESKD patients with DRA, including those detected in amyloid lesions. Direct hemoperfusion utilizing HICBs may play a role in acting Aβ2M amyloidogenesis by reducing the amyloid-related proteins. [ABSTRACT FROM AUTHOR]

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein's aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter's fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo , the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues. Accumulation of β2-microglobin in tissues is responsible for the disease dialysis-related amyloidosis. The D76N mutant is highly amyloidogenic. An in vitro biophysical and spectroscopic investigation of D76N β2-microglobin amyloid fibril formation and its inhibition by the common extracellular chaperone, clusterin, is described. In vivo , clusterin may play an important role in preventing β2-microglobin deposition. CH23082_TOC.jpg [ABSTRACT FROM AUTHOR]

A man in his 20s initiated intermittent peritoneal dialysis in the late 1960s. He subsequently transitioned to hemodialysis and survived for more than 50 years, spanning low-flux and high-flux hemodialysis eras. He underwent surgery for cervical and lumbar spinal canal stenosis after 30 and 35 years, respectively, and both surgeries revealed similar degrees of severe amyloid deposition. At autopsy, significant improvement was seen in lumbar amyloid deposition. During the previous 25 years, serum β2 microglobulin levels had decreased from 40 mg/L and been maintained at 20 mg/L. This case indicates that advances in dialysis therapy aimed at lowering β2 microglobulin concentrations have reduced highly deposited amyloid.

Study Design: Retrospective cohort study.Objective: To develop and evaluate a new grading system for destructive lumbar spondyloarthropathy (DLSA) by assessing bony destruction of the facet joints; to evaluate interrater reliability; and to determine the association between anteroposterior (AP) dural sac diameter at the lumbar level and the new grading.Summary Of Background Data: The characteristics of DLSA are unknown, hindering clinical care and research. Imaging to determine the cause of DLSA may positively contribute to patient outcome or well-being by providing prognostic information.Patients and Methods: In the magnetic resonance images (MRIs), we measured an axial midline AP dural sac diameter and evaluated bone destruction caused by amyloidosis at the level of the center of each lumbar disk of the lumbar spine. Two orthopedic surgeons independently evaluated each case at two-month intervals and assigned the grade by rating bone destruction at each lumbar level. Weighted κ and intraclass correlation coefficients for interrater reliability were calculated. In addition, the correlation between the AP diameter of the spinal dural sac at the lumbar level and the new MRI-based DLSA grade was examined.Results: The sample size of 82 patients was reached by examining records of 118 consecutive patients. The mean (SD) age of the included patients was 65 (7.2) years, and 36 (43.9%) were women. The grading of DLSA showed moderate to good interrater reliability at both assessments (κ, 0.59-0.78). Intraclass correlation coefficient showed substantial to excellent agreement (intraclass correlation coefficient, 0.63-0.86). The AP diameter of the spinal dural sac at the lumbar level showed a significant correlation with the new grading ( P <0.001).Conclusions: The new MRI-based grading system for DLSA has good interrater reliability, although the strength of agreement varies somewhat. The new grading system correlates with AP dural sac diameter. Thus, this classification focused on facet erosion, which leads to functional incompetence may be helpful in surgical decision-making. [ABSTRACT FROM AUTHOR]

Purpose and method: Patients on hemodialysis develop carpal tunnel syndrome (CTS) due to an accumulation of dialysis-related β2 microglobulin (β2m) amyloid (DRA). In Japan, dialysis technology has progressed remarkably in the past 40 years and has increased the time until patients require surgery for CTS. However, unclear is whether the time from the start of hemodialysis to the first surgery for CTS is associated with β2m clearance by the different hemodialysis techniques. Therefore, we retrospectively evaluated β2m clearance, serum β2m levels, and the change in the length of this period in patients across 4 periods according to the year that first surgery for CTS was performed: period 1, 1982–1989; period 2, 1990–1999; period 3, 2000–2009; and period 4, 2010–2019. Result: A total of 222 patients who met the selection criteria were included. Mean β2m clearance was −1.8 ± 16.7% in period 1, and improved to 65.4 ± 8.6% in period 3. Accordingly, the serum β2m value after hemodialysis decreased significantly. The time from the start of hemodialysis to the first surgery for CTS was 12.4 ± 2.9 years in period 1 but increased to 21.8 ± 6.3 years in period 3. In multivariable linear regression analysis, the significant factors contributing to β2m clearance were periods 2, 3, and 4. In particular, the relation between removal of β2m and the extension of the dialysis vintage in period 1 and 2 was remarkable compared with periods 3 and 4. Conclusion: Our findings indicate that improvement of β2m clearance via advances in dialysis technology might result in a significant extension in the time between starting HD and the first surgery for CTS. [ABSTRACT FROM AUTHOR]

Solubility- and supersaturation-limited crystallization and amyloid fibril formation. The cycle of crystallization and dissolution of sodium acetate suggests the formation and dissolution cycle of amyloid fibrils. AFM image of β2-microglobulin amyloid fibrils was modified from Katou et al. Protein Sci. 11(9) (2002) 2218–2229. [Display omitted] • Supersaturation is required for the formation of crystal-like amyloid fibrils. • Breakdown of supersaturation triggers amyloid formation and onset of amyloidosis. • Supersaturation-dependent coupled equilibrium links unfolding and amyloid formation. • Preventing amyloid ignition will be more effective than extinguishing amyloid fire. • Healthy proteostasis involves factors retaining supersaturation of amyloid proteins. From a physicochemical viewpoint, amyloid fibril formation is a phase transition from soluble to crystal-like sates limited by supersaturation. It occurs only above solubility (i.e., the solubility limit) coupled with a breakdown of supersaturation. Although many studies have examined the role of molecular chaperones in the context of proteostasis, the role of supersaturation has not been addressed. Moreover, although molecular chaperone-dependent disaggregations have been reported for preformed amyloid fibrils, amyloid fibrils will not dissolve above the solubility of monomers, even if agitations fragment long fibrils to shorter amyloid particles. On the other hand, on considering a reversible and coupled equilibrium of interactions, folding/unfolding and amyloid formation/disaggregation, molecules stabilizing native states can work as a disaggregase reversing the amyloid fibrils to monomers. It is likely that the proteostasis network has various intra- and extracellular components which disaggregate preformed amyloid fibrils as well as prevent amyloid formation. Further studies with a view of solubility and supersaturation will be essential for comprehensive understanding of proteostasis. [ABSTRACT FROM AUTHOR]

Background Beta-2 microglobulin (β2M) accumulates in hemodialysis (HD) patients, but its consequences are controversial, particularly in the current era of high-flux dialyzers. High-flux HD treatment improves β2M removal, yet β2M and other middle molecules may still contribute to adverse events. We investigated patient factors associated with serum β2M, evaluated trends in β2M levels and in hospitalizations due to dialysis-related amyloidosis (DRA), and estimated the effect of β2M on mortality. Methods We studied European and Japanese participants in the Dialysis Outcomes and Practice Patterns Study. Analysis of DRA-related hospitalizations spanned 1998–2018 (n  = 23 976), and analysis of β2M and mortality in centers routinely measuring β2M spanned 2011–18 (n  = 5332). We evaluated time trends with linear and Poisson regression and mortality with Cox regression. Results Median β2M changed nonsignificantly from 2.71 to 2.65 mg/dL during 2011–18 (P = 0.87). Highest β2M tertile patients (>2.9 mg/dL) had longer dialysis vintage, higher C-reactive protein and lower urine volume than lowest tertile patients (≤2.3 mg/dL). DRA-related hospitalization rates [95% confidence interval (CI)] decreased from 1998 to 2018 from 3.10 (2.55–3.76) to 0.23 (0.13–0.42) per 100 patient-years. Compared with the lowest β2M tertile, adjusted mortality hazard ratios (95% CI) were 1.16 (0.94–1.43) and 1.38 (1.13–1.69) for the middle and highest tertiles. Mortality risk increased monotonically with β2M modeled continuously, with no indication of a threshold. Conclusions DRA-related hospitalizations decreased over 10-fold from 1998 to 2018. Serum β2M remains positively associated with mortality, even in the current high-flux HD era. [ABSTRACT FROM AUTHOR]

Dialysis-related amyloidosis (DRA) is characterized by the deposition of amyloid consisting of beta2- microglobulin in the musculoskeletal system, causing carpal tunnel syndrome, destructive spondyloarthropathy, and/or bone cysts. Increased cystic radiolucency of the bones and tendon thickening due to inflammation are common findings in DRA. We have developed a new dialysis method, extendedhours hemodialysis without dietary restrictions for the aim of improving both hypertension and malnutrition. We retrospectively evaluated the clinical effects of dialysis time on the risk for developing of DRA. The study subjects were all of the 30 patients who had received this treatment for more than 11 years. They were divided into two groups according to the weekly dialysis hours: 15 patients ≥ 16.5 hours/week (L-group) and 15 patients ≤ 15.5 hours/week (S-group). Plain x-ray imaging and ultrasonography were used to assess cystic radiolucency of the bones and thickness/diameter of the soft tissues. The proportion of the carpal bone cystic radiolucency was lower in the L-group. The severity of median nerve compression at the wrist was significantly less in the L-group (right hand: p = 0.0082, left hand: p = 0.0137). Multivariate regression analysis showed that dialysis time was a predictor of median nerve compression (β = -0.559, p = 0.005). In conclusion, extended-hours hemodialysis without dietary restrictions contributes to lower the risk for developing of DRA at the wrist. We therefore propose that extended-hours hemodialysis without dietary restrictions is a preferred method which maintains the patients' quality of life compared with the conventional hemodialysis method. [ABSTRACT FROM AUTHOR]

Till date, there had been no reported case of dialysis-related amyloidosis (DRA) associated with a β2-microglobulin variant. We report here a 41-year-old haemodialysis patient with systemic amyloidosis, exhibiting macroglossia and swelling salivary glands, uncommon clinical manifestations for DRA. Molecular analysis showed that the patient had a new variant of β2-microglobulin (V27M). Extracted amyloid protein was predominantly composed of variant β2-microglobulin. In vitro analysis revealed that this variant β2-microglobulin had a strong amyloidogenic propensity, probably owing to the decreased stability caused by a bulky methionine residue. Our data clearly show that V27M variant is amyloidogenic and this mutation results in unusual clinical manifestations. To date, only one amyloidogenic β2-microglobulin variant (D76N) has been reported in non-dialysis patients. It is noteworthy that the V27M and D76N variants show substantial differences in both clinical phenotypes and pathomechanical features. This is the first case of DRA associated with a naturally occurring β2-microglobulin variant. [ABSTRACT FROM AUTHOR]

Removing β2-microglobulin (β2M) from blood circulation is considered to be the most effective method to delay the occurrence of dialysis-related amyloidosis (DRA). The ideal extracorporeal β2M removal system should be cost-effective, highly specific and having a high capacity. However, the traditional technologies based on size exclusion do not have an adequate specificity, and alternative immunosorbents have limited applications due to low capacity and their high cost. Nanobodies (Nbs), the smallest functional recombinant antibody fragments, offer several advantages to overcome these obstacles. In this study, an anti-β2M Nb with a C-terminal thiol-tag was successfully prepared from E. coli for site-directed and oriented immobilization and usage as capture ligand in a β2M-selective immunosorbent. The prepared immunosorbent showed a high binding capacity of up to 7 mg β2M per mL resin, which is 17 times higher than that of previous studies using single-chain variable antibody fragments (scFv). Furthermore, an exceptional high specificity has been demonstrated as other human serum proteins were not adsorbed during dynamic adsorption experiments. About 80% of the original binding capacity of the immunosorbent was restored after four consecutive easy regenerations, whereas 90% of the original capacity was retained after 1-month storage of the resin. Moreover, the mathematical model fitted very well the in vitro perfusion. The results with this pioneering immunosorbent confirm its possible clinical application and is expected to reach the required clinical effect of immunoadsorption therapy. Dialysis-related amyloidosis (DRA), associated with the accumulation of β2-microglobulin (β2M), is a serious complication of end-stage kidney disease. Removing β2M from blood circulation by extracorporeal blood purification is considered to be the most effective method to delay the occurrence of DRA. However, the existing methods are incapable to eliminate sufficient quantities of β2M from circulation, either because of lack of specificity, high cost or for low capacity. In this manuscript, we provide a practical and economic immunosorbent based on anti-β2M nanobody for DRA. The prepared immunosorbent was reusable and storable, and demonstrated high specificity and realized a high binding capacity of up to 7 mg β2M per mL resin, which is 17 times higher than that of the previous studies. Image, graphical abstract [ABSTRACT FROM AUTHOR]

We report a case of dialysis-related amyloid arthropathy in a patient with end-stage renal disease. It presented in our patient as moderately increased FDG uptake in the amyloid deposition in the periarticular tissues and eroding into adjacent bones.

β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.

The RDT population, initially at 215 patients, exceeded 300,000 in 2011, with a total of 329,609 patients at the end of December 2016. In our Institute, the number of patients with destructive spondylosis is increasing with the increase in the number of dialysis patients in Japan. We had 14 Cases in the 1990s, and then 82 cases in the 2000s and have already had 131 cases in the 2010s. The purpose of this study was to investigate the incidence of dialysis-related amyloidosis (DRA) such as destructive spondyloarthropathy (DSA), dialysis amyloid arthropathy (DAA), and carpal tunnel syndrome (CTS). In addition, another purpose was to examine the risk factors of the DRA. DAA made its own assessment on radiographs based on stage. Survey items were patient's basic data, laboratory data and X-ray view. Patient's basic data included such as sex, age, height, and weight and RDT-related factors such as kidney disease that led to RDT, age at start of RDT, RDT history, medical history (past and present), and history of surgery. The frequency of DRA was examined by medical history and radiological examination in 199 dialysis patients who obtained informed consent. The patients were divided into two groups according to the presence or absence of DRA, and risk factors of DRA were investigated from the medical history, basic data of patients, and blood tests. Of the 199 patients on regular dialysis therapy, 41 (20.6%) showed DRA. Based on the X-ray images, 21 patients (10.6%) showed DSA, while 22 patients (11.1%) showed DAA. Sixteen patients (8.0%) had CTS, determined through a history of surgery. Regarding overlap of conditions, 14 had both DSA and DAA, 3 had both DSA and CTS, and 2 had both DAA and CTS. There were statistically significant differences between the two groups in the cause of disease in Chronic glomerulonephritis and Diabetic Nephropathy, age at the start of RDT, period of RDT, body weight, blood platelet count, and blood Ca level. When multivariate analysis was performed on these items, statistical differences were recognized only during the dialysis period. In conclusion, long dialysis period was a risk factor for DRA. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using β2-microglobulin (β2m), a protein responsible for dialysisrelated amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of β2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged β2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of β2m at several micromoles per liter, a similar concentration range to that in vivo. Since β2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with β2m electrostatically. PolyPs appear to dehydratewater molecules around β2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of β2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis. [ABSTRACT FROM AUTHOR]

β2‐microglobulin‐related (Aβ2M) amyloidosis (dialysis‐associated amyloidosis) is a common complication in long‐term dialysis patients. An increased concentration of β2‐microgloblin (β2‐m) in the serum appears to be a prerequisite for Aβ2M amyloidosis, in turn causing Aβ2M amyloid deposition predominantly in the osteoarticular tissue. There are few reports, however, of Aβ2M amyloid deposition in non‐dialysis patients. We describe an atypical case of a non‐dialysis patient with Aβ2M amyloid deposition in bladder cancer. A Japanese man in his 80s with no history of dialysis was admitted for transurethral resection of bladder cancer. Histopathological analysis revealed a small amount of amyloid deposition in the small‐vessel wall of both the peripheral urothelial carcinoma and necrotic area. Amyloid typing by immunohistochemistry was strongly positive for anti‐β2‐m antibody, and β2‐m was most frequently detected in laser microdissection‐liquid chromatography tandem mass spectrometry. Although Aβ2M amyloidosis was expected, contrary to this, the patient's serum β2‐m was only 4 mg/L, although his urine β2‐m level was increased at 1340 mg/L. The unique findings observed in our patient may contribute to the elucidation of the novel pathogenesis of Aβ2M amyloid fibril formation that is distinct from conventional Aβ2M amyloidosis. [ABSTRACT FROM AUTHOR]

Uremic toxins are linked to chronic kidney disease (CKD)-related systemic diseases. β2-Microglobulin (β2-m), a water-soluble, middle-sized molecule, is associated with mortality and dialysis-related amyloidosis (DRA). DRA occurs in long-term dialysis patients, with β2-m amyloid deposited mainly in osteoarticular tissues. We investigated a model of β2-m amyloid fibril extension at neutral pH in the presence of trifluoroethanol or sodium dodecyl sulfate. Using this model, some biological molecules, including glycosaminoglycans and lysophospholipids, were found to be chaperones for β2-m amyloid fibril extension. Several protein-bound solutes, such as indoxyl sulfate (IS) and p-cresyl sulfate, are independent risk factors for cardiovascular disease in CKD patients, especially those undergoing dialysis. We investigated kidney injury-induced acceleration of atherosclerosis in association with macrophage phenotypic change to a proinflammatory state as well as increased IS deposition in lesions in an animal model. IS directly induced macrophage inflammation and impaired cholesterol efflux to high-density lipoprotein (HDL) in vitro. In addition, a clinical study showed that HDL isolated from CKD patients induced proinflammatory reactions and impaired cholesterol efflux to macrophages. These findings suggest that protein-bound solutes, including IS, will induce dysfunction of both macrophages and HDL in atherosclerotic lesions. To remove uremic toxins efficiently, we demonstrated the potential efficacy of oral charcoal adsorbent and hexadecyl-immobilized cellulose beads in hemodialysis patients. These findings suggest that uremic toxins induce various CKD-related systemic disorders, and further therapeutic strategies will be needed to reduce uremic toxins enough and improve life expectancy in CKD patients. [ABSTRACT FROM AUTHOR]

We report a case of hemodynamic deterioration after aortic valve replacement in a patient with mixed systemic amyloidosis. A 77-year-old male with severe aortic valve stenosis and 19 years hemodialysis underwent aortic valve replacement. Postoperatively, the patient died of hemodynamic deterioration. Autopsy findings showed massive, whole-body edema and mixed systemic amyloidosis (dialysis-related and AA amyloidosis). Clinical and autopsy findings implied that hemodynamic deterioration was caused by increased vascular permeability. The amyloid deposit to the vessel causes inflammatory changes and increases vascular permeability. Mixed systemic amyloidosis occurs very rarely and could increases vascular permeability even more than each single type of amyloidosis. Systemic amyloidosis may be a risk factor for hemodynamic deterioration after cardiac surgery. Patients with longtime hemodialysis and a history associated with dialysis-related amyloidosis would have at least single systemic amyloidosis, which should be considered a contraindication to cardiac surgery with cardiopulmonary bypass. [ABSTRACT FROM AUTHOR]

A 74-year-old woman with a 34-year history of hemodialysis presented with an intermittent fever, which later coincided with recurrent bilateral shoulder and hip joint pain. Imaging studies suggested amyloid arthropathy, which was histologically confirmed by a synovial biopsy. Increasing β 2 -microglobulin clearance during dialysis alone attenuated the intermittent fever and joint pain, but the symptoms did not disappear until the administration of prednisolone 10 mg/day. Reported cases of dialysis-related amyloidosis with a fever imply that changing to blood purification methods with high β 2 -microglobulin clearance is crucial for controlling the condition long-term, whereas concurrent use of anti-inflammatory agents promptly alleviates the symptoms.

This cross-sectional study aimed to determine the utility of ultrasonography with improved resolution using a high-frequency probe for dialysis-related carpal tunnel syndrome (CTS). This study targeted 125 hemodialysis patients at our hospital. A 12 MHz probe was placed on the carpal tunnel area to identify the median nerve. The compression rate of the nerve was calculated by measuring the smallest diameter of the compressed nerve and largest diameter of the unaffected part. To quantify CTS symptoms, we determined the presence of Tinel's sign, measured pinch strength, and used questionnaires to assess numbness and pain. The association of these clinical data with the compression rate was examined. Mean compression rate was 12.1 ± 1.1%. The compression rate cutoff value for those positive with Tinel's sign was 25%, (sensitivity and specificity were 0.80 and 0.91, respectively), and that for those with a history of CTS surgery was 25% (sensitivity and specificity were 0.67 and 0.89, respectively). Multiple regression analysis identified duration of dialysis, β2-microglobulin(β2-Mg) concentration, positivity for Tinel's sign, history of CTS surgery, and pinch strength as independent compression rate determinants. Notably, compression rates were significantly higher in patients with a ≥4-year duration of dialysis and a β2-Mg level of 20 mg/L or more. The compression rate of the median nerve measured by an improved ultrasound device significantly correlated with clinical symptoms, medical history, and serological features associated with dialysis-related CTS. Because ultrasonography is non-invasive, the examination might be a simple method especially for early diagnosis of dialysis-related CTS. [ABSTRACT FROM AUTHOR]

Destructive spondyloarthropathy (DSA) is the most serious spinal complication of dialysis-related amyloidosis in patients on long-term hemodialysis (HD), but we could not find any information about DSA in patients on peritoneal dialysis (PD) for over 10 years. We retrospectively evaluated factors contributing to DSA inHDand PD patients. Sixty-seven patients on dialysis for 10 to 19 years were compared between a PD group (n = 23) or a HD group (n = 44). In the PD group, nine patients (39%) developed DSA.The mean age of DSA patients was significantly higher than that of non-DSA patients (66.2 ± 10.0 vs. 51.0 ± 12.8 years, P = 0.03).The frequency of cervical spine Destructive spondyloarthropathy (DSA) is the most serious spinal complication of dialysis-related amyloidosis in patients on long-term hemodialysis (HD), but we could not find any information about DSA in patients on peritoneal dialysis (PD) for over 10 years. We retrospectively evaluated factors contributing to DSA inHDand PD patients. Sixty-seven patients on dialysis for 10 to 19 years were compared between a PD group (n = 23) or a HD group (n = 44). In the PD group, nine patients (39%) developed DSA.The mean age of DSA patients was significantly higher than that of non-DSA patients (66.2 ± 10.0 vs. 51.0 ± 12.8 years, P = 0.03).The frequency of cervical spine DSA did not show any difference between the PD and HD groups, but the frequency of lumbar spine DSA showed a significant difference (22% vs. 5%, P = 0.04). The serum beta-2 microglobulin (B2MG) level was significantly higher in PD patients than in HD patients (38.4 mg/L vs. 27.4 mg/L, P = 0.0025). Mechanical stress such as elevation of the intra-abdominal pressure due to infusion of PD fluid (1500 mL to 2000 mL) for over 10 years might contribute to lumbar DSA in patients on long-term PD. [ABSTRACT FROM AUTHOR]

Objectives: A β2-microglobulin (β2m) fragment that lacks the first six amino acids, i.e., ΔN6β2-microglobulin (ΔN6β2m), is an endogenous, proteolytically derived, amyloidogenic fragment of β2m, the precursor protein in Aβ2M amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of β2m, in this study we aimed to investigate conformational characteristics of ΔN6β2m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of β2m with C-terminal unfolding (β2m92-99) in serum samples from 10 patients undergoing hemodialysis (HD). Methods: We utilized capillary electrophoretic analysis, surface plasmon resonance and enzyme-linked immunosorbent assay. Results and conclusions: We confirmed the normal core structure of ΔN6β2m with a commercial monoclonal anti-β2m antibody. In addition, using the specific monoclonal antibody for the C-terminal peptide, i.e. mAb 92-99, we confirmed unfolding in the C-terminal region of ΔN6β2m. On the basis of these findings, we established an ELISA to measure β2m92-99 using ΔN6β2m as a standard molecule in circulation. However, we did not detect β2m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum. [ABSTRACT FROM AUTHOR]

Dialysis-related amyloidosis ( DRA) is a unique type of amyloidosis (beta-2 microglobulin) predominantly in end-stage renal disease. Its clinical manifestations add to increased morbidity and reduced quality of life. There seems to be a relative risk reduction in DRA manifestations when hemodialysis ( HD) patients are treated with advanced HD technology, but changes of the course of DRA are uncertain. The aim of our investigation was to evaluate the prevalence and severity of carpal tunnel syndrome ( CTS) in long-term dialysis patients receiving either conventional or high-flux, online-produced ultrapure dialysis fluid. The cross-sectional study included 147 HD patients (at least 10 years). The definitive diagnosis of CTS was made histologically or by the coexistence of CTS with other radiological DRA manifestations (bone cysts, arthropathies). The two HD patient groups did not differ significantly in age at start of HD, gender, major co-morbid diseases, anuria, and dialysis vintage. The conventional HD group had significantly higher circulating beta-2 microglobulin and C-reactive protein ( CRP) levels. The prevalence of DRA was 68% for the conventional HD group and 28% for the advanced HD group. Duration of dialysis treatment was the only significant risk factor for the development of clinical DRA manifestations in both study groups, but CTS, bone cysts, or arthropathies occurred significantly earlier in conventional HD patients. The prevalence and severity of DRA have decreased with advances in dialysis technology during the last two decades, although its occurrence is simply delayed. [ABSTRACT FROM AUTHOR]

To investigate early intermediates of β2-microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X-ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co-crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild-type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation. [ABSTRACT FROM AUTHOR]

We use molecular dynamics simulations of a full atomistic Gō model to explore the impact of selected DE-loop mutations (D59P and W60C) on the folding space of protein human β2-microglobulin (Hβ2m), the causing agent of dialysis-related amyloidosis, a conformational disorder characterized by the deposition of insoluble amyloid fibrils in the osteoarticular system. Our simulations replicate the effect of mutations on the thermal stability that is observed in experiments in vitro. Furthermore, they predict the population of a partially folded state, with 60% of native internal free energy, which is akin to a molten globule. In the intermediate state, the solvent accessible surface area increases up to 40 times relative to the native state in 38% of the hydrophobic core residues, indicating that the identified species has aggregation potential. The intermediate state preserves the disulfide bond established between residue Cys25 and residue Cys80, which helps maintain the integrity of the core region, and is characterized by having two unstructured termini. The movements of the termini dominate the essential modes of the intermediate state, and exhibit the largest displacements in the D59P mutant, which is the most aggregation prone variant. PROPKA predictions of pKa suggest that the population of the intermediate state may be enhanced at acidic pH explaining the larger amyloidogenic potential observed in vitro at low pH for the WT protein and mutant forms. [ABSTRACT FROM AUTHOR]

Dialysis-related amyloidosis is a serious complication of long-term hemodialysis. Its pathogenic mechanism involves accumulation of β2-microglobulin in the blood, which then forms amyloid fibrils and is deposited in tissues, leading to inflammation and activation of osteoclasts. Lixelle, a direct hemoperfusion column for adsorption of β2-microglobulin, has been available since 1996 to treat dialysis-related amyloidosis in Japan. However, previous studies showing the therapeutic efficacy of Lixelle were conducted in small numbers of patients with specific dialysis methods. Here, we report the results of a nationwide questionnaire survey on the therapeutic effects of Lixelle. Questionnaires to patients and their attending physicians on changes in symptoms of dialysis-related amyloidosis by Lixelle treatment were sent to 928 institutions that had used Lixelle, and fully completed questionnaires were returned from 345 patients at 138 institutions. The patients included 161 males and 184 females 62.9 ± 7.7 years age, who had undergone dialysis for 25.9 ± 6.2 years and Lixelle treatment for 3.5 ± 2.7 years. Based on self-evaluation by patients, worsening of symptoms was inhibited in 84.9-96.5% of patients. Of the patients, 91.3% felt that worsening of their overall symptoms had been inhibited, while attending physicians evaluated the treatment as effective or partially effective for 72.8% of patients. Our survey showed that Lixelle treatment improved symptoms or prevented the progression of dialysis-related amyloidosis in most patients. [ABSTRACT FROM AUTHOR]

Background/Aims: Carpal tunnel syndrome (CTS) is a common clinical presentation of dialysis-related amyloidosis. It was determined whether β2-microglobulin (β2M) and advanced glycation end products in serum are predictors of CTS in dialysis patients. Methods: A total of 385 hemodialysis patients were screened for CTS. β2M in serum was determined by a competitive enzyme-linked immunoassay, CML by a competitive enzyme-linked immunosorbent assay and total pentosidine by reverse-phase high-performance liquid chromatography. Results: 127 patients (33%) were treated with biocompatible membranes, 174 (45%) with high-flux dialysis. 122 patients (31.7%) had clinical signs of CTS. Significant predictors of CTS were: age, female gender, serum β2M, total protein, dialysis with non-biocompatible high-flux dialysis compared to non-biocompatible low-flux dialysis, Kt/V and serum concentration of CML (OR 2.47 for the 3rd vs. 1st quartile, 95% CI 1.229-4.961, p = 0.011). Conclusion: The prevalence of CTS as a possible manifestation of dialysis-related amyloidosis is still high. Serum concentration of CML may be a predictor of CTS besides β2M and malnutrition. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We previously identified an intermediate β2-microglobulin (I-β2m), which is an amyloidogenic β2m variant, via capillary electrophoresis (CE) and reported hemodialysis (HD)-associated variations in the serum concentrations of each β2m component, including that found in the rebound phase. Recent research has indicated that I-β2m can bind, via the SO3- moiety, with glycosaminoglycan or proteoglycan, which are major components of interstitial tissue. Because alterations in I-β2m are likely to be important in view of the possible accumulation of amyloidogenic precursor proteins in the interstitial space, we studied the I-β2m profile as related to HD. We used CE to determine the I-β2m profile both at the start and at the end of HD and during the rebound phase in 12 HD patients. We found both an unfolded β2m and a destructured I-β2m. More important, two peaks appeared in the rebound phase, one suggesting a refolding and one suggesting an irreversible destruction. Given that the intercompartmental transfer coefficient for β2m is 1.0, our results indicated concomitant processes occurring after HD: refolding of the β2m conformation and trapping of destructured I-β2m in the extravascular space. Because the trapping of destructured I-β2m supposedly leads to accumulation of β2m in the interstitial space, we have proposed a new concept-a 'shuttle' concept-for amyloid formation from β2m in the HD setting. [ABSTRACT FROM AUTHOR]

Background/Aims: The aim of this study was to determine whether treatment with β2-microglobulin adsorption column (Lixelle) affects bone cysts and clinical symptoms in patients with dialysis-related amyloidosis (DRA). Methods: Radiographic changes in the number and area of bone cysts of the wrist and the hip joint were compared between 39 hemodialysis (HD) patients treated with Lixelle (Lixelle group) and 28 HD patients treated with conventional therapy as retrospective control (HD group). Clinical symptoms of DRA were also evaluated. Results: In the Lixelle group, the number of bone cysts and the cystic area in wrist joints were significantly decreased, although the changes in these parameters in hip joints were not significant. In the HD group, the corresponding parameters in the hip joints even significantly increased. Clinical symptoms notably improved after Lixelle treatment. Conclusion: Treatment with Lixelle reduces the radiolucency of bone cysts in the wrist joints, and improves clinical symptoms associated with DRA. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Carpal tunnel syndrome occurs frequently in long-term hemodialysis patients. However, the literature contains few detailed reports of other nerve entrapment syndromes of the upper extremity in these patients. We encountered 4 cases in which cubital tunnel syndrome occurred in long-term hemodialysis patients. In all cases, a hypertrophic synovial mass projecting from the humeroulnar joint compressed the ulnar nerve, and Congo red staining revealed that the mass contained amyloid deposition. Synovial proliferation resulting from amyloid arthropathy of the elbow joint appears to be the primary cause of this disease. [ABSTRACT FROM AUTHOR]

Beta-2 microglobulin (β2m) is the light chain of Class I major histocompatibility complex (MHC-I) complex. β2m is an intrinsically amyloidogenic protein capable of forming amyloid fibrils in vitro and in vivo. β2m displays the typical immunoglobulin-like fold with a disulphide bridge (Cys25–Cys80) cross-linking the two β-sheets. Engineering of the loop comprised between β-strands D and E has shown that mutations in this region affect protein structure, fold stability, folding kinetics and amyloid aggregation properties. Such overall effects have been related to the DE loop backbone structure, which presents a strained conformation in the wild-type (wt) protein, and a type I β-turn in the W60G mutant. Here, we report a biophysical and structural characterization of the K58P-W60G β2m mutant, where a Pro residue has been introduced in the type I β-turn i + 1 position. The K58P-W60G mutant shows improved chemical and temperature stability and faster folding relative to wt β2m. The crystal structure (1.25 Å resolution) shows that the Cys25–Cys80 disulphide bridge is unexpectedly severed, in agreement with electrospray ionization–mass spectrometry (ESI–MS) spectra that indicate that a fraction of the purified protein lacks the internal disulphide bond. These observations suggest a stabilizing role for Pro58, and stress a crucial role for the DE loop in determining β2m biophysical properties. [ABSTRACT FROM PUBLISHER]

Removal of β2-microglobulin (β2M) from the blood of patients suffering from kidney dysfunction is crucial to protect those individuals from getting the diseased state of dialysis-related amyloidosis. By harnessing the nucleation-dependent fibrillation process of amyloidogenesis, a β2M removal strategy has been proposed by preparing seed-conjugated polymer beads and assimilating soluble β2M to the fibrils on the surface at neutral pH. A novel peptide segment of β2M ranging from residue 58 to residue 67 (Lys-Asp-Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr), which was capable of being fibrillated at neutral pH was isolated. Charge interaction between the positive N-terminal lysine and the negative C-terminal α-carboxylic group was demonstrated to be critical for the molecular self-assembly leading to the peptide fibril formation by favoring β-sheet conformation. Because the peptide fibrils were successful to seed intact β2M at neutral pH, the fibrils were immobilized on polymer beads of HiCore resins, and the resulting seed-conjugated beads were used to accrete intact β2M in the form of fibrils elongated on the bead surface. Its efficiency of the β2M removal was improved by placing the seed-immobilized beads in the middle of a continuous flow of the β2M-containing solution as practiced in the blood circulation during the hemodialysis. Therefore, this β2M removal system is suggested to exhibit high specificity, high binding capacity, and cost-effectiveness appropriate for eventual clinical application to remove β2M from the blood of renal failure patients. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 [ABSTRACT FROM AUTHOR]

Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of β2-microglobulin (β2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of β2m (ΔN6β2m). The crystal structure of ΔN6β2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops—corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation—are unmasked and fold into a new two-stranded antiparallel β-sheet. The β-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular β-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid fibril elongation in denatured proteins involves cycles of coupled binding and misfolding. To gain insights into possible kinetic intermediates, we performed hydrogen/deuterium exchange of amide protons during fibril elongation with β2-microglobulin (β2-m) at pD =2.5, under which conditions β2-m is acid denatured. To study the conformational change in monomeric β2-m monitored by NMR spectroscopy, we used 15N-labeled monomers and nonlabeled seeds. Pulse-labeling hydrogen/deuterium exchange with a quenched-flow apparatus indicated that the rate-limiting intermediate at pD =2.5 is not protected from the exchange, even disrupting a hydrophobic cluster present in the acid-denatured β2-m. Significant protection was acquired upon transition to the fibrils. In view of the suggestion that the rate-limiting intermediates are bound to the lateral surface of seed fibrils, weak interactions with a largely unfolded conformation might be useful for their dynamic sliding to the growing ends. The results support a new model of fibril elongation with intermediates bound to the lateral surface of seeds. [ABSTRACT FROM AUTHOR]

Abstract: Renal failure impairs the clearance of β2-microglobulin from the serum, with the result that this protein accumulates in joints under the form of amyloid fibrils. While the molecular mechanism leading to deposition of amyloid in vivo is not totally understood, some organic compounds, such as trifluoroethanol (TFE), are commonly used to promote the elongation of amyloid fibrils in vitro. This article gives some insights into the structural properties and the conformational states of β2-microglobulin in the presence of TFE, using both the wild-type protein and the mutant Trp60Gly. The structure of the native state of the protein is rather insensitive to the presence of the alcohol, but the stability of this state is lowered in comparison to some other conformational states. In particular, a native-like folding intermediate is observed in the presence of moderate concentrations of TFE. Instead, at higher concentrations of the alcohol, the population of a disordered native-unlike state is dominant and correlates with the ability to elongate fibrils. [Copyright &y& Elsevier]

Abstract: Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β2-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid fibrils elongate seed dependently, with preformed fibrils providing a template for propagation of amyloidogenic conformation. Most seeding experiments use relatively few seed fibrils in comparison with monomers, resembling steady-state enzyme kinetics. Pre-steady-state kinetics should also be useful for characterizing the elongation process. With β2-microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, we measured the pre-steady-state kinetics of fibril elongation at pH 2.5, conditions under which the monomer is largely unfolded. β2-m has Trp residues at positions 60 and 95. We used three single Trp mutants and fluorescence spectroscopy to study structural change upon fibril elongation. To focus on conformational change in monomers, we prepared seeds with a mutant without a Trp residue. At a fixed concentration of monomeric β2-m, the apparent rate of fibril elongation increased with an increase in the concentration of seeds and then saturated, suggesting the accumulation of a rate-limiting intermediate. Importantly, saturation occurred at a seed/monomer ratio of around 10, as expressed by the concentration of the monomer. Because the number of monomers constituting the seed fibrils is much larger than 10, the results suggest that the elongation process is limited by “non-active-site binding.” Spectral analysis indicated that, upon this non-active-site binding, both Trp60 and Trp95 are exposed to the solvent, and then only Trp60 is buried upon transition to the fibrils. We propose a new model of fibril elongation in which non-active-site binding plays a major role. [Copyright &y& Elsevier]

Patients on long-term hemodialysis can develop dialysis-related amyloidosis (DRA) due to deposition of β2-microglobulin (β2m) into amyloid fibrils (Aβ2M). Despite intensive biochemical studies, the pathogenesis of amyloid deposition in DRA patients remains poorly understood. To elucidate the mechanisms that underlie Aβ2M fibril formation in DRA, we generated transgenic mice that overexpress human β2m protein in a mouse β2m gene knockout background (h B2MTg+/+ m B2m+/+). The h B2MTg +/+m B2m−/− mice express a high level of human β2m protein in many tissues as well as a high plasma β2m concentration (192.8 mg/L). This concentration is >100 times higher than that observed in healthy humans and >4 times higher than that detected in patients on dialysis. We examined spontaneous and amyloid fibril-induced amyloid deposition in these mice. Amyloid deposition of β2m protein was not observed in aged or amyloid fibril injected animals. However, mouse senile apolipoprotein A-II amyloidosis (AApoAII) was detected, particularly in the joints of mice that were injected with AApoAII amyloid fibrils. This study demonstrates that this mouse model could be valuable in studying the components and conditions that promote DRA, and indicates that high plasma concentrations of hβ2m as well as seeding with pre-existing amyloid fibrils may not be sufficient to induce Aβ2M. [ABSTRACT FROM AUTHOR]

β2-microglobulin (β2m) is capable of forming amyloid in osteoarticular structures in kidney failure patients that undergo chronic hemodialysis treatment. Although sophisticated analytical methods have yielded comprehensive data about the conformation of the native protein both as a monomer and as the light chain of the type I major histocompatibility complex, the cause and mechanisms leading to the transformation of β2m into amyloid deposits in patients with dialysis-related amyloidosis are unsettled. The impact on conformational stability of various truncations, cleavages, amino acid substitutions, and divalent cations, especially Cu2+, however, are highly relevant for understanding β2m unfolding pathways leading to amyloid formation. This review describes the current knowledge about such conformationally destabilizing and amyloidogenic factors and links these to the structure and function of β2m in normal physiology and pathology. Tables listing modifications of β2m found in amyloid from patients and a systematic overview of laboratory conditions conducive to β2m-fibrillogenesis are also included. [ABSTRACT FROM AUTHOR]

β2-Microglobulin (β2m) forms amyloid fibrils in patients undergoing long-term haemodialysis, leading to dialysis-related amyloidosis. Proteolysis of the N-terminal region of β2m results in a truncation of the six N-terminal residues (ΔN6 β2m) in ∼30% of the β2m molecules extracted from ex vivo fibrils. The ΔN6 β2m has been shown to exhibit a higher tendency for self-association comparing to the wild-type (wt) β2m, particularly at neutral pH. In order to gain atomic insights into the early stages of amyloid formation of the wt and ΔN6 β2m, various molecular dynamics simulations were conducted to investigate the stability and dynamics of these two molecules at various temperatures and neutral pH in this study. Our results, in agreement with previous experimental results, indicate that the structural stability of the ΔN6 β2m is lower than that of the wt β2m. It can be attributed to fact that the removal of the N-terminal six residues results in the loss of the salt-bridge interaction between residues R3 and D59, leading to the increased solvent exposure of the K3 peptide. It further allows water molecules to destabilise the interior region of the K3 peptide, leading to the elongation between the B- and E-strands. It may further accelerate the conformational changes of the ΔN6 β2m, leading to the formation of amyloid fibrils more readily at neutral pH. Our results also suggest that the K3 peptide may be a potential initiation site of amyloid formation for the ΔN6 β2m due to its increased solvent exposure. We further suggest that fibril morphology of the ΔN6 β2m formed at neutral pH is similar to that of the wt β2m formed at low pH (1.5-3) since they adopt the similar conformation with the elongation between B- and E-strands for their partially unfolded amyloidogenic intermediates. [ABSTRACT FROM AUTHOR]

Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteinsto form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorterfibrils. Here, combining these 2 opposing effects on β2-microglobulin (132-m). a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of ≈1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis. [ABSTRACT FROM AUTHOR]

Abstract: β2-microglobulin (β2m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β2m are still largely unknown. In vitro, acidic conditions, pH<4.5, induce amyloid fibrillation of native β2m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β2m—found in the circulation of many dialysis patients—is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β2m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β2m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β2m. This may explain the apparent lack of detectable amounts of the variant β2m in extracts of amyloid from DRA patients. [Copyright &y& Elsevier]

Dialysis-related amyloidosis is related to the inefficient removal of β2-microglobulin (β2-m) that is mainly responsible for the formation of amyloid fibrils deposited on the joints and in the heart, blood vessels and digestive system. Magnetically assisted hemodialysis (MAHD) can be used for the prevention of dialysis-related amyloidosis. MAHD is based on ferromagnetic nanoparticle-targeted binding substance conjugates (FN-TBS Cs) that should be administered to the patient before the dialysis session. The TBS should have a high affinity for β2-m so that the conjugates bind with the β2-m in the bloodstream. The complex FN-TBS–β2-m will be selectively removed during dialysis by means of a “magnetic dialyzer” that is installed at the dialysis machine in series to the conventional dialyzer. We have examined the in vitro applicability of MAHD by employing biocompatible Fe3O4 and bovine serum albumin (BSA) as constituents of the FN-TBS Cs. We evaluated the binding capacity of both bare Fe3O4 FNs and Fe3O4-BSA Cs for β2-m concentrations ranging from mild to severe conditions. Finally, we conducted mock-dialysis experiments for the evaluation of several technical issues related to MAHD. β2-m is adsorbed onto the Fe3O4-BSA Cs not only almost instantly, but also very efficiently. The employed Cs do not chemically interact with the materials used in standard dialyzers, as agglomerates were not observed in the capillaries of the conventional dialyzers. MAHD may become an efficient modality for the prevention of dialysis-related amyloidosis because β2-m concentrations ranging from mild to severe conditions can be adequately handled. [ABSTRACT FROM AUTHOR]