HUMAN MICROVASCULAR ENDOTHELIAL TISSUE-CULTURE CELL MODEL FOR STUDYING PATHOGENESIS OF BRAZILIAN PURPURIC FEVER

Brazilian purpuric fever (BPF) is a fulminant pediatric disease charac terized by fever, with rapid progression to purpura, hypotensive shock , and death. All known BPF eases have been caused by three clones of H aemophilus influenzae biogroup aegyptius and have occurred in either B razil or Australia. Using an immortalized line of human vascular endot helial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F . D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W . Ades, J. Infect Dis. 169:430-433, 1994). With multiplicities of infe ction (MOIs) as low as one bacterium per 1,000 tissue culture cells, B PF-associated strains produce a unique cytotoxic effect in which the t issue culture cells detach and aggregate in large floating masses afte r 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which prod uce the cytotoxic phenotype were able to replicate intracellularly whe reas non-BPF-associated strains, with MOIs of greater than or equal to 1,000 did not replicate and did not produce the phenotype. We also sh owed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacteria l cell fractions at MOIs of >1,000 produced the cytotoxic effect. Furt hermore, bacteria in numbers equal to MOIs of >1,000 treated with chlo ramphenicol did not produce the cytotoxic phenotype, suggesting a requ irement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-mu m-pore-size me mbrane also failed to produce the phenotype. The ability of the bacter ium to invade, replicate, and produce the phenotype appears to be prim arily parasite directed since phagocytosis, pinocytosis, and eukaryoti c protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to i nvade and cause a cytotoxic response. Understanding the basic mechanis ms involved in this tissue-destructive process should enhance our know ledge of the general pathogenesis of BPF.