Biomedical subjects

T A Gaither

Publications and source records attributed to T A Gaither.

27 records · Page 2Linked to original sources

PubMed1983

The interaction of C3b bound to pneumococci with factor H (beta 1H globulin), factor I (C3b/C4b inactivator), and properdin factor B of the human complement system.

Pneumococcal cell walls are potent activators of the alternative complement (C) pathway; pneumococcal capsules are not. C3b that is deposited onto the cell walls of encapsulated organisms, however, functions inefficiently in host defense compared to C3b deposited onto capsular polysaccharides. Results of previous studies with guinea pig erythrocytes suggested that C3b deposited onto surfaces that do not activate the alternative pathway is rapidly inactivated. In the present study, we examined the interactions of C3b bound to pneumococcal capsules, to pneumococcal cell walls, and to the surface of sheep erythrocytes (E) with the serum control proteins, Factor H (beta 1H globulin) (H) and Factor I (C3b/4b inactivator) (I), and with Factor B (B) of the alternative C pathway. Conversion of bound C3b to C3bi was assayed by binding of radiolabeled conglutinin in a quantitative binding assay. Neither pneumococcal cell wall C3b nor capsular C3b was converted efficiently to a conglutinin-binding form by serum incubation. Experiments with purified C components showed that, after incubation with H and I, fewer conglutinin-binding sites were created on pneumococci than on E bearing equal numbers of C3b. Molecular analysis demonstrated that this did not result from cleavage of pneumococcal-bound C3b to an unusual, nonconglutinin-binding form of the molecule. Binding studies in which radiolabeled H was used demonstrated that the majority of C3b that is bound to both pneumococcal capsules and cell walls bound H with a lower affinity than did E-bound C3b. Studies of the binding of radiolabeled B demonstrated that C3b that was bound to pneumococcal cell walls and to E demonstrated equal affinity for B. In contrast, the majority of C3b that was fixed to pneumococcal capsules bound B with only 1/30 as high affinity. We conclude that pneumococcal capsules are not alternative pathway activators because the low affinity of capsular C3b for B leads to inefficient formation of an alternative pathway convertase, C3bBb. With regard to H binding, both cell wall- and capsular-bound C3b act as if they were in a "protected site" and resist degradation by the control proteins.

Binding Sites↗

PubMed1983

Complement receptor expression by neoplastic and normal human cells.

Complement receptor (CR) expression in cell lines derived from Burkitt's lymphoma (BL), Epstein Barr virus-transformed cord blood lymphocytes (CB), and peripheral lymphocytes from patients with infectious mononucleosis (IM) was examined. Red cell intermediates bearing various densities of C4b, C3b, C3bi, or C3d were tested for rosette formation with the cell lines. In addition, a series of studies was performed under conditions that precluded the cleavage of cellbound C3b by Factor I (C3bINA). These conditions did not alter rosetting by the cells that were tested. RAJI cells rosetted with EAC3bi greater than EAC3d greater than EAC3b, but not with EAC4b. EAC3b/RAJI rosette formation required much greater quantities of C3b bound to red cells than did CB and IM lines, which unlike RAJI, also bound EAC4b. All of the BL lines failed to bind EAC4b even at a C4b density of 50,000 molecules/cell, but several lines did form rosettes with EAC3b, and most formed rosettes with EAC3bi and EAC3d. Fluid phase C3b blocked RAJI/EAC3b rosetting while having little effect on RAJI/EAC3bi rosette activity. Moreover, fluid phase C3b, as well as C4b, blocked RAJI/EAC3b rosettes more effectively than CB/EAC3b rosettes. The results indicate that the RAJI cell line has a receptor for C3b, with characteristics that differ markedly from the C3b receptor of cell lines derived from CB lymphocytes and of lymphoblastoid cell lines derived from patients with IM. This receptor is capable of interacting with soluble but not cellbound C4b. In these studies, rosette formation was examined under various ionic conditions. RAJI/EAC3b rosette formation was severely reduced as ionic strength was increased, whereas RAJI/EAC3bi binding was only moderately decreased at physiologic ionic strength. In striking contrast, EAC3bi binding to monocytes, PMN, and human erythrocytes was markedly reduced as ionic strength increased, but EAC3b binding to these cells was less sensitive to changes in ionic strength. Under conditions of physiologic ionic strength, the C3bi receptor of phagocytic cells may be at a functional disadvantage in the binding of C3bi-coated particles. This may have major physiologic implications.

Burkitt Lymphoma↗

PubMed1982

Biotinylation of human C3.

Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.

Avidin↗

PubMed1982

The use of conglutinin in a quantitative assay for the presence of cell-bound C3bi and evidence that a single molecule of C3bi is capable of binding conglutinin.

We have developed a quantitative assay for cell surface C3bi using 125I-labeled conglutinin. Conglutinin was purified to homogeneity from bovine serum and radiolabeled with 125I Bolton Hunter reagent. Conditions of time, temperature, ionic strength, and cell concentration that optimized the binding of conglutinin to erythrocytes bearing C3bi were then determined. The interaction between conglutinin and C3bi under these conditions was highly specific, since EAC4b3b, EAC4b3d, EAC4b3b-beta IH, and EAC4b treated with serum did not bind radioconglutinin significance better with EA or EAC4b. Using this assay, was examined the kinetics of inactivation of both human and guinea pig C3b bound to erythrocytes and showed that, for both, maximum conglutinin binding occurred after EAC4b3b had been incubated with a source of beta 1H and C3INA for 10 to 20 min at 37 degrees C.l We showed a linear relationship between the number of molecules of C3bi per erythrocyte and the amount of conglutinin bound for both guinea pig and human C3bi. The affinity of conglutinin for cell-bound C3bi was shown to be independent of C3bi density on the erythrocyte surface, and the Kd for conglutinin binding to erythrocytes bearing human C3bi was determined to be 1.3 X 10(-8) M. The number of conglutinin binding sites per erythrocyte as calculated from Scatchard plots was equal to the number of C3bi molecules on the cell surface as determined by direct assay using 125I-labeled C3. Moreover, for both human and guinea pig C3bi, the plot of log (cell surface C3bi) vs log (conglutinin bound) had a slope of 1. These findings imply that a single molecule of C3bi is capable of binding a molecule of conglutinin under the conditions of our assay.

Animals↗

PubMed1981