Blocking immunoglobulin G (IgG) inhibits complement-mediated killing of serum-resistant Neisseria gonorrhoeae (GC) in immune human serum. We examined the mechanism of action of blocking IgG. Presensitization of GC with increasing concentrations of blocking IgG or F(ab')2 before incubation with bactericidal antibody and absorbed pooled normal human serum increased consumption and deposition of the third component of human complement (C3) and the ninth component of human complement (C9) but inhibited killing in dose-related fashion. We next showed that blocking IgG or F(ab')2 partially inhibited binding of bactericidal IgG to GC. Also, binding of a monoclonal antibody recognizing GC outer membrane protein PIl was almost completely inhibited by blocking F(ab')2, confirming other work (Rice, P. A., M. R. Tam, and M. S. Blake, manuscript submitted for publication) showing that PIl is a target for blocking antibody. Studies of the C3 deposition site showed that one quarter of the C3 deposited on GC in the presence of blocking IgG bound covalently to the antibody molecule. Finally, 125I-GC constituents with covalently bound C3 were affinity purified on Sepharose bearing antibodies to C3 and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. C3 deposition on a 40,000-mol wt surface protein was enhanced six-to ninefold by blocking IgG, which indicates that the site of complement deposition was altered by blocking antibody. These studies show that blocking IgG competes with bactericidal antibody for binding to GC, but enhances rather than blocks complement activation, and leads to complement deposition at new sites that do not result in serum killing.

Regional delivery of drugs can offer a pharmacokinetic advantage in the treatment of localized tumors. One method of regional delivery is by intra-arterial infusion into the basilar/vertebral artery network that provides local access to infratentorial tumors, which are frequent locations of childhood brain cancers. Proper delivery of drug by infused solutions requires adequate mixing of the infusate at the site of infusion within the artery lumen. Our mixing studies with an in vitro model of the vertebral artery network indicate that streaming of drug solution is likely to occur at low, steady infusion rates of 2 ml/min. Streaming leads to maldistribution of drug to distal perfused brain regions and may result in toxic levels in some regions while concurrently yielding subtherapeutic levels in adjacent regions. According to our model findings, distribution to both brain hemispheres is not likely following infusion into a single vertebral artery even if the infusate is well-mixed at the infusion site. This outcome results from the unique fluid flow properties of two converging channels, which are represented by the left and right vertebral branches converging into the basilar. Fluid in the model remains stratified on the side of the basilar artery served by the infused vertebral artery. Careful thought and planning of the methods of intravertebral drug infusions for treating posterior fossa tumors are required to assure proper distribution of the drug to the desired tissue regions. Improper delivery may be responsible for some noted toxicities or for failure of the treatments.

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Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.

Gram-positive cocci resist direct killing by serum. The mechanism of resistance was studied by measuring consumption of terminal complement components from serum and uptake of purified, radiolabeled C7 and C9 on rough and encapsulated type 7 Streptococcus pneumoniae. Extensive consumption of C5, C7, and C9 occurred when 5 X 10(8) rough or type 7 pneumococci were incubated for 1 hr in 10% pooled normal human serum (PNHS). Approximately 10,000 molecules of C7 and C9 bound per organism during the same period of incubation. Twenty to 30% of C7 and C9 was released from rough organisms. Release was not due to autolysis since it occurred with glutaraldehyde-fixed organisms as well as in S. pneumoniae that were rendered resistant to autolysis by growth in ethanolamine. Between 10 and 30% of bound 125IC9 counts were eluted from the rough and type 7 organisms by incubation in 1 M NaCl or 0.01 M EDTA, which suggests that bound C5b-9 was not attached by predominantly ionic interactions. Elution of 44 to 74% of 125IC9 from live and glutaraldehyde-fixed organisms by 1% sodium deoxycholate suggests that hydrophobic bonds are involved in C5b-9 attachment. Trypsin cleaved 67 and 55% of 125IC9 counts from live rough and type 7 S. pneumoniae, respectively which indicates that the bound complex is not protected by the cell wall from proteolytic attack. Serum resistance in S. pneumoniae does not represent a failure to form C5b-9 on the bacterial cell wall but apparently reflects a failure of the bound complex to penetrate the thick peptidoglycan layer.