Ryu, Kyu-Eun
(Department of Biomedical Sciences Catholic University)
,
Rhim, Hyang-Shuk
(Department of Biomedical Sciences Catholic University)
,
Park, Chong-Won
(Division of Hematology, Department of Internal Medicine, College of Medicine, Catholic University)
,
Chun, Heung-Jae
(Department of Biomedical Sciences Catholic University)
,
Hong, Seung-Hwa
(Blood Products Division, Biologics Evaluation Department, Korea Food and Drug Administration)
,
Kim, Young-Chai
(School of Chemical Engineering, College of Engineering, Hanyang University)
,
Lee, Young-Moo
(School of Chemical Engineering, College of Engineering, Hanyang University)
In a continuation of our previous studies on blood compatibility profiles of anion-substituted poly(vinyl alcohol) (PVA) membranes, in which hydroxyl groups have been replaced with carboxymethyl (C-PVA) and sulfonyl groups (S-PVA), we have studied the activation of complement components and the chan...
In a continuation of our previous studies on blood compatibility profiles of anion-substituted poly(vinyl alcohol) (PVA) membranes, in which hydroxyl groups have been replaced with carboxymethyl (C-PVA) and sulfonyl groups (S-PVA), we have studied the activation of complement components and the changes in white cell and platelet count in vitro and compared them with those of unmodified PVA, Cuprophane, and low-density polyethylene. Complement activation of fluid phase components, C3a, Bb, iC3b, and SC5b-9, and of bound phases, C3c, C3d, and SC5b-9, were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot, respectively. The changes in the number of white cells and platelets following complement activation were counted using a Coulter counter. C-PVA and S-PVA activated C3 to a lesser extent than did PVA, which we attribute to the diminished level of surface nucleophiles of the samples. In addition, C- and S-PVA exhibit increased inhibition of Bb production, resulting in a decrease in the extent of C5 activation. Consequently, because of the reduced activation of C3 and C5, C- and S-PVA samples cause marked decreases in the SC5b-9 levels in plasma. We also found that the negatively charged sulfonate and carboxylate groups of the samples cause a greater extent of adsorbtion of the positively charged anaphylatoxins, C3a and C5a, because of strong electrostatic attraction, which in turn provides an inhibition of chemotaxis and activation of leukocytes. The ability to inhibit complement production, together with the binding ability of anaphylatoxins of the C- and S-PVA samples, leads to a prominent decrease in lysis of leukocytes as well as activation of platelets.
In a continuation of our previous studies on blood compatibility profiles of anion-substituted poly(vinyl alcohol) (PVA) membranes, in which hydroxyl groups have been replaced with carboxymethyl (C-PVA) and sulfonyl groups (S-PVA), we have studied the activation of complement components and the changes in white cell and platelet count in vitro and compared them with those of unmodified PVA, Cuprophane, and low-density polyethylene. Complement activation of fluid phase components, C3a, Bb, iC3b, and SC5b-9, and of bound phases, C3c, C3d, and SC5b-9, were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot, respectively. The changes in the number of white cells and platelets following complement activation were counted using a Coulter counter. C-PVA and S-PVA activated C3 to a lesser extent than did PVA, which we attribute to the diminished level of surface nucleophiles of the samples. In addition, C- and S-PVA exhibit increased inhibition of Bb production, resulting in a decrease in the extent of C5 activation. Consequently, because of the reduced activation of C3 and C5, C- and S-PVA samples cause marked decreases in the SC5b-9 levels in plasma. We also found that the negatively charged sulfonate and carboxylate groups of the samples cause a greater extent of adsorbtion of the positively charged anaphylatoxins, C3a and C5a, because of strong electrostatic attraction, which in turn provides an inhibition of chemotaxis and activation of leukocytes. The ability to inhibit complement production, together with the binding ability of anaphylatoxins of the C- and S-PVA samples, leads to a prominent decrease in lysis of leukocytes as well as activation of platelets.
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문제 정의
ESCA Studies. Since the aim of this study is to examine the eifects of substituted anions to PVA on the activation of complement components, the confirmation of substitution of test samples is a matter of importance. In this study, the presence of the anionic substitution of the modified PVAs was confirmed by using ESCA.
Our previous studies revealed that anion substituted poly(vinyl alcohol) membranes, carboxymethylated and sulfonated poly(vinyl alcohol) membranes, possess improved anti-coagulation activities, solute permeabilities, and inhibition properties of plasma protein adsorption due to substitutions.710 The aim of this study is to examine the effects of these substitutions on complement activation. The present paper reports the blood compatibility profile of the anion substituted cross-linked poly(vinyl alcohol) membranes characterized by the activation of complement together with the changes in white cell and platelet counts.
제안 방법
Immunoassays of Complement Activation. Complement components of fluid phase were analyzed by using commercially available ELISA kits (C3a, Bb, iC3b, and CS5b-9; C3a EIA, Bb fragment EIA, iC3b EIA, and SC5b-9 EIA; Quidel, USA, respectively, and C5a; EIA C5a micro; Behring Diagnostics, Germany). Complement components bound to the surface were evaluated by immunoblot analysis of eluted complements using monoclonal antibody for SC5b-9 (Quidel, USA) and polyclonal antibodies for C3c and C3d (Dako, Denmark).
Since the aim of this study is to examine the eifects of substituted anions to PVA on the activation of complement components, the confirmation of substitution of test samples is a matter of importance. In this study, the presence of the anionic substitution of the modified PVAs was confirmed by using ESCA. Figure 1 shows the ESCA survey scan spectra of PVA, S-PVA, and C-PVA.
성능/효과
In conclusion, the substitution of hydroxyl groups with anion lead to a marked decrease in complement activation. The diminished level of surface nucleophiles of C- and S- PVA activate less extent of C3 than PVA.
In other words, LDPE produced the least amount of C3a and C3b, whether produced C3b bound to Bb or converted into iC3b. Therefore, the result that LDPE produced the least amount of iC3b among samples is of little importance, but the fact that it produced much lower amount of Bb than iC3b is of great importance. Another interesting aspect of samples on C3 activation is that C-PVA exhibited increased inhibition activity of Bb compared to S-PVA, though both C- and S-PVA samples have a diminished level of nucleophiles due to substitution.
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