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Biomedical subjects

J Janatova

Publications and source records attributed to J Janatova.

16 recordsLinked to original sources
PubMed1992

Adsorption of complement proteins on surfaces with a hydrophobicity gradient.

Activation of the complement system is recognized as one of the major problems with respect to biocompatibility of biomaterials. The binding of C3 (central component of complement) and B (factor B, an activator of C3), and H (factor H, an inhibitor of C3 activation) plays a crucial role in the activation of the alternative pathway of complement on the surfaces of biomaterials during extracorporeal procedures. Here we report on the adsorption of C3, B or H on to the silica surface with a hydrophobicity gradient. The amount of native 125I-C3 bound to both hydrophilic and hydrophobic surfaces was very similar (0.8 and 0.9 micrograms/cm2; 4 x 10(-12) mol/cm2). Neither factor H nor factor B was able to displace already adsorbed 125I-C3 from either of the surfaces. The extent of binding of factors B and H to preadsorbed C3 was a function of the surface hydrophobicity: more 125I-B or 125I-H was bound to C3 adsorbed at the hydrophilic end than at the hydrophobic end of the gradient surface. The binding of 125I-B or 125I-H to preadsorbed C3 appeared to be influenced by the availability of their binding sites on adsorbed C3 molecules rather than by the amount of surface-bound C3. At the hydrophobic end of the gradient surface the molar binding ratio of B/C3 was considerably smaller than the molar binding ratio of H/C3. It can be speculated that the hydrophobicity of the surface determines orientation and/or conformation of adsorbed C3 molecule; when adsorbed at the hydrophobic end of the gradient, C3 molecule predominantly exposes the binding site to which only factor H can bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1992

Modulation of complement activation on hemodialysis membranes by immobilized heparin.

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

Biocompatible Materials↗
PubMed1991

Biomedical polymers differ in their capacity to activate complement.

Conventionally, complement activation by biomedical polymers has been evaluated by determining the C3a concentration in the fluid phase only. According to this criterion, biomaterials such as hemodialysis membranes made from cellulosic or various synthetic polymers were classified as activators or nonactivators of complement. Since certain membranes bind large quantities of C3a from the fluid phase, classification based on fluid-phase C3a concentration has in some instances been inaccurate. As follows from the comparison of complement activation by cuprophane and polyacrylonitrile membranes, the capacity of a biomedical polymer to activate complement is not determined by the number of potential covalent binding sites on its surface. Biomaterial itself may lack hydroxyl and/or amino groups, and yet it may activate C3 in human serum very efficiently. Some of the biomaterials may also bind unactivated/unfragmented C3 whether in the absence or presence of other serum proteins. In addition, binding of factor B (a promotor of C3 activation) and binding of factor H (an inhibitor of C3 activation) to certain biomaterials have been found to be independent of complement activation and unaffected by the presence or absence of C3. Thus, it is becoming apparent that the requirements for the formation and stability of the C3 convertase on artificial surfaces differ from those on biological membranes, and that the relative magnitude of binding of factor B and factor H to the surface per se cannot be used as a reliable indicator of the capacity of the biomaterial to activate complement. Further studies are necessary to elucidate the molecular mechanisms of C3 and C5 activation on the surfaces of biomedical polymers.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Proteins↗
PubMed1990

Activation of complement by hemodialysis membranes: polyacrylonitrile binds more C3a than cuprophan.

Conventionally, complement activation by hemodialysis membranes has been determined by measuring fluid phase C3a. Based on such measurements, polyacrylonitrile (PAN) membranes have been classified as weak activators compared to cuprophan. Previous studies have demonstrated, however, that PAN adsorb fluid phase C3a. Based on that observation, we hypothesized that complement activation by PAN might be artifactually underestimated if relatively large amounts of C3a remained membrane bound. In the present study, a method that allows the simultaneous quantification of both fluid phase and membrane bound C3a was used to assess complement activation by PAN and cuprophan. Pieces of membrane were incubated with C3-depleted serum that had been repleted with radiolabeled C3. Subsequently, the supernates and membranes were subjected to SDS-PAGE, and complement activation was quantified by determining the radioactivity of the C3a bands in the gel. The results showed that while the serum exposed to cuprophan membranes contained almost five times more C3a than that exposed to PAN, approximately 80 times more C3a was bound to the PAN membranes. Consequently, the total amount of C3a generated in the presence of PAN was higher than that generated in the presence of cuprophan. We conclude that assessment of complement activation by hemodialysis membranes using fluid phase C3a measurements alone may be misleading.

Acrylic Resins↗
PubMed1989

Disulfide bonds are localized within the short consensus repeat units of complement regulatory proteins: C4b-binding protein.

Several plasma and membrane proteins belong to a superfamily of structurally related proteins that contain internal homology of a variable number (2-30) of repeating units. Each SCR (short consensus repeat) unit is approximately 60 amino acid residues in length, with the positions of 1 Trp, 2 Pro, and 4 Cys residues being conserved. The aim of this study was to provide experimental evidence that each SCR may exist as an independent structural domain maintained by disulfide bonds. The well-characterized C4b-binding protein (C4BP) with eight SCR units in each of its seven identical chains was chosen for this study. Analysis of the disulfide-bonding pattern indicated that intrachain disulfide bonds may be localized within each SCR unit, with the first and third and the second and fourth half-cystines in each unit being linked. This pattern of disulfides may confer to C4BP (and to other structurally related proteins) a conformation which apparently allows the assembly of the SCR units (4-30) in a tandem fashion. Such an arrangement of the polypeptide chain(s) may explain, in part, the elongated shape of these protein molecules. The structural motif of the SCR units of C4BP is discussed in relation to those previously described for the type II domain of fibronectin and the kringle structure present in various proteins of the coagulation system.

Amino Acid Sequence↗
PubMed1989

Activation of the alternative pathway of complement by cellulosic hemodialysis membranes.

Compared to cellulose acetate, hemodialysis with cuprophan membranes is associated with greater activation of the alternative pathway of complement. Previous studies have shown that this difference is not due to a greater number of potential covalent binding sites for activated C3 on cuprophan. To investigate further the factors that influence complement activation by hemodialysis membranes, proteins were eluted from serum-treated cuprophan and cellulose acetate membranes with hydroxylamine at alkaline pH and analyzed by SDS-PAGE and Western blot. Approximately 23 times more total protein was removed from cellulose acetate. Virtually all the C3 in the cellulose acetate eluate was in the form of inactive fragments C3c and C3dg. In contrast, the functionally active form of C3 (C3b) was a prominent constituent of the cuprophan eluate. The binding of factor B (precursor of the catalytic subunit of the C3 convertase) and factor H (regulatory protein of C3 activation) to serum-treated membranes was also analyzed. By Scatchard's method, the affinity constant at equilibrium for factor B binding (KB) to the two types of membranes was not significantly different; however, there were approximately four times more factor B binding sites on the cuprophan than on the cellulose acetate. For cuprophan, the number of factor B binding sites was 1.6 times greater than the number of factor H binding sites. These studies demonstrate that a portion of the C3b molecules that bind to cuprophan are protected from degradation, and suggest that the complement activating capacity of hemodialysis membranes is determined by biochemical properties that modulate both the binding of serum proteins to the membrane and the interactions of the endogenous regulatory proteins with membrane-associated C3b.

Cellulose↗
PubMed1989

Analysis of the complement C3 fragments associated with hemodialysis membranes.

During hemodialysis with cuprophan membranes, bioactive peptides are generated because the alternative pathway of complement is activated. When cellulose acetate membranes are employed, complement activation is attenuated. The molecular basis for this improved biocompatibility is unknown. It has been postulated, however, that the complement activating potential of dialysis membranes is influenced by the availability of free hydroxyl groups which would provide an acceptor site for activated C3. To investigate this hypothesis, the forms of C3 associated with cellulose acetate and cuprophan membranes have been analyzed. By Western blot, the predominant form of C3 present on both types of membranes is C3c, a degradation product of C3 that lacks the thiolester necessary for covalent bonding. Minimal amounts of C3d (the region of C3 which contains the thiolester) were observed on both membranes; however, by ELISA, there was no difference in the amount bound to cellulose acetate compared to cuprophan. Further, membrane-associated C3d could be removed by urea, suggesting that it was not bound covalently. These studies indicate that the complement activating potential of dialysis membranes is not determined primarily by the availability of potential covalent binding sites for activated C3b.

Blotting, Western↗
PubMed1988

Neutrophil mobilization induced by complement fragments during experimental group B streptococcal (GBS) infection.

Degradation products of the third component of complement have been reported to have the ability to mobilize leukocytes from the marrow and induce leukocytosis. The effect of C3d,g preparations on neutrophil responses in a neonatal rat model of group B streptococcal infection in which neutrophil mobilization from the marrow is inadequate has been evaluated. Dimeric and monomeric fragments of C3d,g were isolated from human serum; the identity of the C3d,g preparations was confirmed by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing. Uninfected neonatal rats responded to intraperitoneal injection of C3d,g with a peripheral blood neutrophilia at 30 minutes and 4 hours after inoculation. C3d,g, which lacks intrinsic chemotactic activity, enhanced the local accumulation of neutrophils in the peritoneal cavity of infected, but not uninfected, neonatal rats. In addition, myeloid cell release from the marrow of isolated femurs of neonatal rats receiving C3d,g was significantly enhanced. Thus, the effect of C3d,g in this model was to mobilize marrow cells and induce peripheral leukocytosis. Chemotactic factors released at the site of infection then resulted in the local accumulation of these inflammatory cells. Complement-derived components capable of releasing marrow myeloid elements may play a major role in determining the outcome of bacterial infection in the immature host.

Animals↗
PubMed1987

Activation of complement in human serum by some synthetic polymers used for intraocular lenses.

Determination of the potential to activate complement can be used as one criterion in testing the biocompatibility of various synthetic polymers that are utilized in the medical field. Intraocular lenses (IOLs) made of poly(methyl methacrylate) (PMMA) with PMMA loops, poly(hydroxyethyl methacrylate) (PHEMA) lenses, silicone lenses, and PMMA lenses with polypropylene loops were examined in this study. The concentrations of the activation peptides C3a, C4a and C5a were measured by radioimmunoassay (r.i.a.) in human serum after incubation with and without IOLs for up to 12 h. The presence of silicone lenses caused an increase in C3a levels. In the presence of polypropylene loops, the concentrations of both C3a and C5a were significantly higher than in serum incubated alone. There was no statistically significant increase in the concentration of C4a caused by any of the materials tested. The results suggest that IOLs made from silicone or lenses with polypropylene loops activate the complement system via the alternative pathway.

Biocompatible Materials↗
PubMed1986

Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement.

Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.

Complement C3↗
PubMed1985

Activation and fragmentation of the third (C3) and the fourth (C4) components of complement: generation and isolation of physiologically relevant fragments C3c and C4c.

The degree of the activation and fragmentation of C4 and C3, including chain structure of the activation products, was evaluated by SDS-PAGE analysis of the C4 or C3 antigens that were withdrawn from the reaction media with appropriate immunoadsorbent beads. Full activation of C4 and C3, and subsequent quantitative conversion of C4b into C4c, and C3b into iC3b took place in fresh NHS after the activation of complement with both aggIgG and CVF. For complete conversion of iC3b to C3c erythrocytes carrying the C3b receptor were added to the already activated serum. Both C4c and C3c were isolated by a 2-step procedure involving (i) an adsorption to and (ii) electrophoretic desorption from the respective immunoadsorbent beads.

Complement Activation↗
PubMed1984

Defective binding of the third component of complement (C3) to Streptococcus pneumoniae in multiple myeloma.

Patients with multiple myeloma (MM) are at an increased risk for infections with bacteria that require opsonization with complement. Because Streptococcus pneumoniae is the most frequently encountered pathogen in these patients, we investigated the ability of serum from patients with MM to mediate the binding of C3b, the major opsonin of the complement system, to S. pneumoniae. S. pneumoniae types 3, 14, and 25 were chosen for study, since S. pneumoniae type 3 activates primarily the classical complement pathway (CCP), type 25 primarily the alternative complement pathway (ACP), and type 14 both pathways. S. pneumoniae were treated with normal serum or serum from 17 patients with MM, and the bound C3b was quantified with fluorescein-conjugated anti-C3 in a spectrophotofluorometric assay. Despite normal or elevated serum concentrations of C3, total hemolytic complement, and C-reactive protein in all of the MM sera, factor B in 16/17 such sera, and C4 in 14/17 MM sera studied, all 17 sera demonstrated a defect in C3b binding to type 3 (32.7% +/- 6% of normal). In addition, serum from 15/17 patients bound decreased amounts of C3b to types 14 (39.6% +/- 8%) and 25 (52.2% +/- 8%). Mixing normal serum with MM serum restored MM C3b binding activity to all three S. pneumoniae types, suggesting that the defect was related to a deficiency rather than an inhibitor of C3 activation. Although MM patients are unable to produce specific antibodies to bacterial antigens, the addition of anti-S. pneumoniae antibodies to MM serum did not enhance C3b binding to any of the S. pneumoniae types. However, when S. pneumoniae were opsonized in a mixture of MM serum and C3-depleted normal serum, C3b binding was restored to all three S. pneumoniae types, demonstrating that MM C3 functions normally in the presence of other normal serum factors. In the present studies, the MM C3b binding defect appeared to correlate with the incidence of S. pneumoniae infections. Serum from patients with a history of an S. pneumoniae infection bound significantly less C3 (20.5% +/- 4%) than those study patients without a history of an S. pneumoniae infection (55.8% +/- 8%) (p less than 0.0025). Thus, MM serum has a defect in the activation of C3, and this may contribute to the increased susceptibility of MM patients to S. pneumoniae infections.

Complement C3↗
PubMed1982

Third component of human complement: localization of the internal thiolester bond.

Human complement protein C3 was inactivated by using methylamine and thereby generating a SH group from the internal thiol ester. The protein was coupled via this SH group to activated thiol-Sepharose and digested with elastase. Fragment C3d remained attached to the thiol-Sepharose and was subsequently eluted with L-cysteine. Concomitantly, the original SH group was regenerated, and it was then labeled with iodo[2-(3)H]acetic acid. Partial sequence analysis of the radiolabeled C3d fragment showed that both components of the thiol ester are located close to the amino terminus (residues 23 and 26). Specific chemical cleavage of the alpha-chain was achieved after S-cyanylation of the thiol. The two fragments obtained corresponded to the amino-terminal section (M(r) approximately 46,000) and the carboxy-terminal section (M(r) approximately 70,000). These results together indicate that fragment C3d occupies approximately positions 345-610 of the alpha-chain. The partial sequence of C3d was extended by completion of the sequence of a previously described tryptic peptide. Comparison of residues 1-49 of C3d with a peptide from alpha(2)-macroglobulin [Swenson, R. P. & Howard, J. B. (1980) J. Biol. Chem. 255, 8087-8091] shows a previously recognized identity of seven residues around the thiol ester site and a second region of identity around a known glycosylation site of alpha(2)-macroglobulin. The relationships among these proteins and protein C4 are discussed. An overall outline of the structure of C3 is presented, showing the locations of various fragments and cleavage sites. The thiol ester group places constraints on the local folding of the peptide chain; a possible conformation is suggested and discussed in relation to the mechanism of activation.

Amino Acid Sequence↗