Yoshinori Uji

A Circulating β2-Microglobulin Intermediate in Hemodialysis Patients

Background/Aims: A misfolded β₂-microglobulin (β₂m) is a principle component in dialysis-related amyloidosis. However, no such conformational variant of β₂m has yet been reported in a clinical setting. Capillary electrophoresis is a tool that can identify the conformational variant of β₂m. Methods: Capillary electrophoresis was used to measure a transitional intermediate from native β₂m (N-β₂m) to the amyloid β₂m. This technique was utilized to assay for intermediate β₂m (I-β₂m) in serum from 31 hemodialysis (HD) patients before and after HD, 5 patients with non-dialysis chronic renal failure (CRF), and 5 healthy persons. Results:The predialysis values of serum I-β₂m and N-β₂m were 2.7 ± 1.4 and 29.4 ± 6.8 mg/l, respectively, in the HD patients. The presence of serum I-β₂m correlated weakly with the total serum β₂m concentration in all HD patients. The serum N-β₂m concentration decreased significantly during two types of dialysis treatment: by 32.8% on HD using a polymethylmethacrylate (PMMA) membrane and by 71.2% on online hemodiafiltration (HDF) with a polysulfone (PS) membrane. On the other hand, a dialysis-associated change in serum I-β₂m varied from –36.4 to +203.5% in HD patients using PMMA and from –70.8 to +62.5% in online HDF patients using PS. Moreover, a rebound β₂m profile suggested that I-β₂m might be immobilized in the extracellular space. Conclusion: This study demonstrated that two or three conformational isomers of β₂m were probably ubiquitously recognized in human serum. Though no progressive increase in serum I-β₂m concentration could be found along with HD, this study shows a significantly poor removal of I-β₂m in comparison to N-β₂m in patients receiving ongoing dialysis treatment, even with online HDF.

Hyperdry human amniotic membrane is useful material for tissue engineering: Physical, morphological properties, and safety as the new biological material

Human amniotic membrane (AM) has been used widely as graft biomaterial for a variety of clinical applications. But, there are some persistent problems related to the preparation, storage, and sterilization. To resolve these problems, we developed hyperdry AM (HD-AM) using far-infrared rays, depression of air, and microwaves and then sterilized by γ-ray irradiation. To elucidate the benefit of HD-AM as biological materials, compare with the physical and histological properties of HD-AM with a freeze-dried AM (FD-AM) as typical freeze-dried methods, evaluate the safety of HD-AM in vivo experiment used nude mice, and demonstrate the feasibility of HD-AM transplant in pterygium. The water permeability and the sieving coefficient of HD-AM were significantly lower than that of FD-AM. HD-AM has kept the morphological structure of epithelium and connective tissues. At 18 months after transplanted, single and multilayers of HD-AM in the intraperitoneal cavity was degraded without any infiltrated cells. For clinical treatment, recurrence of pterygium and regrowth of the subconjunctival fibrosis were not observed during the 6-month follow-up periods after the surgery. It was proposed that HD-AM was a safe and effective new biological material for clinical use including treatment for recurrent pterygium.

C-terminal unfolding of an amyloidogenic β2-microglobulin fragment: Δn6β2-microglobulin

Abstract 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.

Effect of heparin on conformation of the β2-microglobulin molecule.

Heparin, one of the essential molecules called glycosaminoglycans (GAGs), is the anticoagulant that is commonly used in regular hemodialysis, during which dialysis‐related amyloidosis (DRA) may develop. The pathogenic protein, i.e. precursor protein, in DRA is β2‐microglobulin (β2m). Recent studies defined amyloidosis as a protein misfolding disease of precursor proteins including β2m. Because the analytic technique capillary electrophoresis can identify molecular variants of the folded β2m, i.e. conformational variants, we utilized it to investigate the effect of heparin on β2m conformation and thus determined whether heparin can promote DRA development by inducing a conformational change in the amyloidogenic β2m molecule. Heparin had a slight but significant effect on intermediate β2m conformation but no effect on native β2m conformation and on conversion of native to intermediate β2m. Our findings thus suggest a possible association of β2m with GAGs containing a sulfate moiety, including heparin, in HD patients.

Capillary Electrophoretic Profile of β2‐Microglobulin Intermediate Associated With Hemodialysis

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.