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.

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.

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.

Rapid optimization of immunoadsorbent characteristics.

Immunoaffinity chromatography is employed in many research areas. We have developed an assay system that overcomes some of the tediousness and uncertainty in dealing with immunoadsorbents (IA). The preparation of IA and the effect of various procedures on the dissociation of antigen-antibody complex and the regeneration of IA are rapidly screened by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Non-covalently bound proteins are dissociated and separated during the electrophoresis from IA beads. A wide variety of associative and dissociative conditions can be tested on small amounts of IA. Information about biospecifially and non-biospecifically adsorbed proteins can also be obtained. By using relatively small volumes of media containing either an antigen or another biospecific molecule, the optimal parameters for affinity chromatography (specificity, binding capacity, efficiency of solvents in dissociation of the complex and their effect on the adsorbent), or even for ion-exchange chromatography, can be determined without first performing several time- and material-consuming chromatographic experiments.

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.