Tetsunosuke Kunitomo

A new form of amyloid protein associated with chronic hemodialysis was identified as β2-microglobulin

Amyloid fibrils were isolated from amyloid-laden tissue obtained from a chronic hemodialysis patient with carpal tunnel syndrome. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous low molecular weight fraction. The protein was found to be identical to beta 2-microglobulin, with regard to its molecular weight of 11,000, amino acid composition and 16 amino-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin.

An enzyme-linked immunosorbent assay for the measurement of human interleukin-6

An enzyme-linked immunosorbent assay has been developed to measure human interleukin-6. The assay, based on the avidin-biotin amplified two-step sandwich method, is quick (requiring 4.5 h), sensitive (detecting 9.5 pg/ml) and satisfactory in reproducibility and specificity. It shows good correspondence with the results of bioassays, and it is not affected by serum and plasma components. These results indicate that this ELISA is suitable for application to clinical samples, which is a major advantage over the widely used bioassays.

Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lys171 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.

Ocular inserts for controlled release of antibiotics

Ocular inserts impregnated with antibiotics (erythromycin and erythromycin estolate) which have sustained release characteristics were prepared, mainly for the purpose of trachoma therapy. In vitro experiments showed that the elution rate of a drug with low solubility in water (erythromycin estolate) is constant when the water content of the hydrogel insert is more than 30%. In the case of a drug with higher solubility (erythromycin), the elution rate depends on the water content. Some in vivo experiments using rabbit eyes were also reported.

Inhibitory effects of β2-microglobulin on invitro calcification of osteoblastic cells

Elevated levels of aluminum and β 2 -microglobulin have been demonstrated in chronic dialysis patients. The role of aluminum in the pathogenesis of renal osteodystrophy has also been shown. We report on the effects of β 2 -microglobulin on calcification in vitro using osteoblastic cells, clone MC3T3-E1. At concentrations comparable to those in plasma of chronic dialysis patients, both β 2 -microglobulin and aluminum suppressed calcification while collagen synthesis and alkaline phosphatase activity were maintained. These observations may be related to the impaired bone mineralization frequently observed in chronic dialysis patients.

A Sensitive Serodiagnosis of Hepatitis C Virus (HCV) Infection with Two Non‐Fused Peptides: Comparison of Antibody Responses Detected with a Newly Developed Assay and a Commercial Second‐Generation Test

An enzyme‐linked immunosorbent assay (ELISA) was developed for the detection of anti‐HCV antibody. We assayed for antibodies against either oligopeptide (S29‐1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false‐positive results induced by non‐specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non‐fused S4 peptide was prepared by the recombinant DNA technique and site‐specific proteolysis (by factor Xa). In 71 non‐A, non‐B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29‐1/S4 ELISA as well as by a second‐generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti‐N14 (core) and C100‐3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29‐1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti‐S29‐1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non‐fused protein (S4) by recombinant DNA technique and a combination of S29‐1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.

Development of a New Blood Purifing System Using Antithrombogenic Materials.

By combining a polyacrylonitrile (PAN)-polyethyleneoxide (PEO) membrane with ionically heparin-bound catheter, tubing, and module header, a totally antithrombogenic continuous ultrafiltration system (ACUS) was developed. Antithrombogenicity of PAN-PEO hollow fiber membrane was considered to be due to the presence of highly concentrated PEO near the inner surface of the membrane wich has a finely dispersed microstructure. ACUS was clinically applied to 24 patients without systemic anticoagulation and one PAN-PEO filter functioned for average 32 hrs without deteriorating their bleeding tendencies.

Permselectivity of ionic solutes across charged dialysis membranes: in vitro and in vivo diffusive mass transfer

Abstract To elucidate the effect of ionic functional groups incorporated into dialysis membranes on the diffusive mass transfer of ionic solutes across those membranes, poly(methyl methacrylate) (PMMA) hollow fiber membranes with ionic functional groups were evaluated in in vitro and in vivo settings and compared with a non-ionic cellulosic membrane as a control. Enhanced iontransfer across a membrane with a functional group having an opposite charge to that of the ion (s) transferred was demonstrated under certain conditions in in vitro experiments. Similarly, reduced transfer was observed when the charge on the ionic function group was of the same sign as the ion(s) transferred. A theoretical model based upon the assumption of Donnan equilibrium and a linear concentration gradient inside the membranes, was proven to predict those characteristic patterns of ion-transfer experimentally demonstrated. In clinical settings, similar enhanced or suppressed ion-transfer was observed under concentration gradients of the ions involved and was reflected in the blood levels of those ions in patients for a limited number of cases such as bicarbonate and acetate ions during hemodialysis with an acetate dialysate.

Evaluation of hemofilter Dynamic behavior of middle molecules (MM) in hemofiltration (HF)

最近の血液浄化法の傾向として, 従来の小分子量物質除去の目的から一歩前進して, 中・大分子量物質の除去が注目されている. それに伴って種々のタイプの人工腎臓が開発されているが, 今回, これらの中からRP-6 (PAN膜), B1-L (PMMA), PAN-15 (PAN膜) の濾過型人工腎臓 (以下Hemofilter) を選び, Hemofiltration (以下HF) における低分子量蛋白, β2-microglobulin (以下β2-MG), myoglobin (以下Mb) の透過性能及び除去性能について検討した. RP-6, BI-Lではβ2-MGは除去され, RP-6では透過性が認められたが, BI-Lではsieving coefficient (以下SC) とは相関しない動きが見られた. すなわち, β2-MGはPMMA膜に吸着されることを示唆しているものと考えられた. PAN-15ではβ2-MGはHFによりHctやTPの変化からみた血液濃縮以上の上昇を示し, 生体内で何んらかの反応が起こっている可能性を示唆しているが, 今回の検討でははっきりしなかった. 今後の課題として免疫学的な検討をも必要とする可能性を残したものといえる. Mbの動きについてはβ2-MGとほぼ同じ変化を示しているが, RP-6, BI-Lでは透過性は微少なものであり, PAN-15ではほとんど認められず, むしろ濃度の上昇すら見られた.