Influence of heparin molecular size on the induction of C- terminal unfolding in ß2-microglobulin.

Dialysis-related amyloidosis (DRA) is characterized by accumulation of amyloid ß2- microglobulin (ß2m) in the interstitial matrix. Matrix substances such as heparin have reportedly been strongly implicated in the pathogenesis of dialysis-related amyloidosis. In clinical setting of hemodialysis, two types of heparin, i.e., high and low molecular heparin (H.M.H. and L.M.H.) have been routinely used. Still commonly used is H.M.H., followed by L.M.H. preparations with distinct advantages. Here, we studied that the interaction of native and two amyloidogenic ß2m variants: ΔN6ß2m and D76N ß2m with H.M.H. and L.M.H. We also investigated whether heparin could induce ß2m to have an amyloidogenic conformation. Biolayer interferometry revealed that ΔN6ß2m had a strong reaction and D76N ß2m had a moderate reaction with H.M.H.. Furthermore,H.M.H. induced the C-terminal unfolding in a native ß2m. By contrast, L.M.H. showed no reaction even with ΔN6ß2m. This study showed firstly a direct binding of ß2m with H.M.H.. H.M.H. would provoked a C-terminal unfolding of ß2m, which indicated production of an amyloidogenic intermediate, i.e., ß2m92-99. In addition, our findings also suggest that L.M.H. may provide beneficial effects against the development of the DRA.

C-terminal unfolding of an amyloidogenic ß2-microglobulin fragment: ΔN6ß2-microglobulin.

OBJECTIVES: A ß2-microglobulin (ß2m) fragment that lacks the first six amino acids, i.e., ΔN6ß2-microglobulin (ΔN6ß2m), is an endogenous, proteolytically derived, amyloidogenic fragment of ß2m, the precursor protein in Aß2M amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of ß2m, in this study we aimed to investigate conformational characteristics of ΔN6ß2m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of ß2m with C-terminal unfolding (ß2m92-99) 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 ΔN6ß2m with a commercial monoclonal anti-ß2m 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 ΔN6ß2m. On the basis of these findings, we established an ELISA to measure ß2m92-99 using ΔN6ß2m as a standard molecule in circulation. However, we did not detect ß2m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum.

Increased levels of small dense low-density lipoprotein cholesterol associated with hemorheological abnormalities in untreated, early-stage essential hypertensives.

Among subfractions of low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (SdLDL-C) has been highlighted as the most atherogenic lipoprotein cholesterol. The present study aimed to compare the relationship of SdLDL-C with blood viscosity, a surrogate marker for cardiovascular disease, with that of other lipid fractions with blood viscosity in essential hypertensives (EHTs). In 128 untreated, early-stage EHTs, blood viscosity was measured with a falling-ball microviscometer, and serum levels of lipid fractions were determined. Blood and plasma viscosity was significantly higher in 49 patients with dyslipidemia (fasting serum level of LDL-C > 140 mg dl(-1), triglyceride > 150 mg dl(-1) or high-density lipoprotein cholesterol (HDL-C) < 40 mg dl(-1)) compared with 79 patients without dyslipidemia, although hematocrit and RBC rigidity index 'k' did not differ between the two groups. Together, SdLDL-C, LDL-C, triglyceride and large LDL-C were positively correlated with blood viscosity, but for HDL-C, the correlation was negative. After adjusting for non-lipid variables that correlated with blood viscosity (that is, the age, body mass index, resting diastolic blood pressure, sex, hematocrit, plasma viscosity and homeostasis model of assessment of insulin resistance), SdLDL-C was most strongly associated with blood viscosity among the lipid fractions. These data suggest that SdLDL-C could strongly increase blood viscosity in EHTs.

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.

Capillary electrophoretic profile of ß2-microglobulin intermediate associated with hemodialysis.

We previously identified an intermediate ß(2)-microglobulin (I-ß(2) m), which is an amyloidogenic ß(2) m variant, via capillary electrophoresis (CE) and reported hemodialysis (HD)-associated variations in the serum concentrations of each ß(2) m component, including that found in the rebound phase. Recent research has indicated that I-ß(2) m can bind, via the SO(3)(-) moiety, with glycosaminoglycan or proteoglycan, which are major components of interstitial tissue. Because alterations in I-ß(2) m are likely to be important in view of the possible accumulation of amyloidogenic precursor proteins in the interstitial space, we studied the I-ß(2) m profile as related to HD. We used CE to determine the I-ß(2) m 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 ß(2) m and a destructured I-ß(2) m. 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 ß(2) m is 1.0, our results indicated concomitant processes occurring after HD: refolding of the ß(2) m conformation and trapping of destructured I-ß(2) m in the extravascular space. Because the trapping of destructured I-ß(2) m supposedly leads to accumulation of ß(2) m in the interstitial space, we have proposed a new concept-a "shuttle" concept-for amyloid formation from ß(2) m in the HD setting.

Effect of heparin on conformation of the ß2-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 ß(2)-microglobulin (ß(2)m). Recent studies defined amyloidosis as a protein misfolding disease of precursor proteins including ß(2)m. Because the analytic technique capillary electrophoresis can identify molecular variants of the folded ß(2)m, i.e. conformational variants, we utilized it to investigate the effect of heparin on ß(2)m conformation and thus determined whether heparin can promote DRA development by inducing a conformational change in the amyloidogenic ß(2)m molecule. Heparin had a slight but significant effect on intermediate ß(2)m conformation but no effect on native ß(2)m conformation and on conversion of native to intermediate ß(2)m. Our findings thus suggest a possible association of ß(2)m with GAGs containing a sulfate moiety, including heparin, in HD patients.

[Genetic diagnostics of pathogenic splicing abnormalities in the clinical laboratory--pitfalls and screening approaches].

In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

[Effect of ISO15189 accreditation in Clinical Laboratory Center of Toyama University Hospital].

Quality, cost, risk, and knowledge control are essential in clinical laboratory management. In addition, a benchmark set by a third party is important. However, it is very difficult to develop these systems without a guideline and an authoritative body to set the benchmark. ISO15189 issued in 2003 is an international standard of "medical laboratories--particular requirements for quality and competence". This international standard is superior as a guideline. ISO15189 specifies quality and competence particular to medical laboratories. Also, it can be used by medical laboratories to develop their quality management systems and assess their own competence, and by accreditation bodies to confirm or recognize the competence of medical laboratories. In Japan, the Japan Accreditation Board for Conformity Assessment (JAB) is an accreditation body of ISO15189. We were awarded ISO15189 accreditation on July 31, 2008. In this report, we summarize the preparations for ISO15189 accreditation and the effects on our laboratory.

A circulating beta 2-microglobulin intermediate in hemodialysis patients.

BACKGROUND/AIMS: A misfolded beta(2)-microglobulin (beta(2)m) is a principle component in dialysis-related amyloidosis. However, no such conformational variant of beta(2)m has yet been reported in a clinical setting. Capillary electrophoresis is a tool that can identify the conformational variant of beta(2)m. METHODS: Capillary electrophoresis was used to measure a transitional intermediate from native beta(2)m (N-beta(2)m) to the amyloid beta(2)m. This technique was utilized to assay for intermediate beta(2)m (I-beta(2)m) 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-beta(2)m and N-beta(2)m were 2.7 +/- 1.4 and 29.4 +/- 6.8 mg/l, respectively, in the HD patients. The presence of serum I-beta(2)m correlated weakly with the total serum beta(2)m concentration in all HD patients. The serum N-beta(2)m 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-beta(2)m 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 beta(2)m profile suggested that I-beta(2)m might be immobilized in the extracellular space. CONCLUSION: This study demonstrated that two or three conformational isomers of beta(2)m were probably ubiquitously recognized in human serum. Though no progressive increase in serum I-beta(2)m concentration could be found along with HD, this study shows a significantly poor removal of I-beta(2)m in comparison to N-beta(2)m in patients receiving ongoing dialysis treatment, even with online HDF.

Marked increases in macrophage colony-stimulating factor and interleukin-18 in maintenance hemodialysis patients: comparative study of advanced glycation end products, carboxymethyllysine and pentosidine.

BACKGROUND: Recent studies have demonstrated the crucial involvement of advanced glycation end products (AGEs) in major complications of long-term hemodialysis (HD) patients. HD, in a clinical setting, is characterized by increased production of proinflammatory cytokines. AGEs and cytokines are presumed to be responsible for the development of major complications in long-term HD. We therefore investigate here the relationship between a newly identified cytokine, interleukin-18 (IL-18), and two AGEs, carboxymethyllysine-hemoglobin (CML-Hb) and pentosidine. METHODS: CML-Hb, pentosidine macrophage colony-stimulating factor (M-CSF), and IL-18 were evaluated in 35 patients undergoing stable maintenance HD. CML-Hb and pentosidine were measured by a dot blot and competitive ELISA. Cytokines were measured with a cytokine-specific ELISA. RESULTS: Circulating levels of CML-Hb and pentosidine were elevated in HD patients as compared to controls. The serum values of M-CSF and IL-18 were significantly increased in the HD patients in comparison to controls. Moreover, these two AGEs and serum values of M-CSF, M-CSF and IL-18 showed significant correlation by simple and multiple regression analysis. CONCLUSION: Elevation of circulating IL-18 levels was demonstrated in maintenance HD patients relative to controls. A correlative increase in M-CSF and IL-18 suggests the presence of a primed state of monocytes/macrophages in HD patients.

Separation of human serum transferrins with different iron-binding states by high-performance liquid chromatography using a pyridinium polymer column.

Four molecular forms of transferrins with different iron-binding states were separated by HPLC using a pyridinium polymer column. The elution order was monoferric transferrin bound to the C-site, holotransferrin, apotransferrin and monoferric transferrin bound to the N-site. Human sera were also analyzed with the column, and ICP-MS combined with HPLC was used to detect iron in each peak. Transferrin peaks separated by HPLC were also confirmed by an immunological method. The percentages of iron saturation in transferrins obtained by the HPLC method were compared with the values calculated from clinical data.