CpG ODN stimulates a TH1 response through its receptor Toll-like receptor 9 (TLR9). TLR9 is a receptor that is found intracellularly. Microparticles are efficiently internalized by dendritic cells (DCs) and macrophages and would thus be an ideal delivery vehicle for CpG ODN to reach its target site thereby enhancing the TH1 response to an antigen also encapsulated in the microparticle. Here, we show that careful control over fabrication parameters can produce biodegradable microparticles with predictable size distributions, surface morphology, and shape. Entrapment efficiencies of the model antigen OVA ranged from 19% to 23% with an average loading of 10 microg/mg of microparticles. For CpG ODN, these values were 33% to 35%, which corresponded to an average loading of 8.5 microg/mg of microparticles. The microparticles release CpG ODN and OVA in a burst followed by sustained release profile. At the highest concentration of microparticles incubated with a pure DC cell line, 92% of DCs had internalized microparticles by 16 hours, confirming that DCs efficiently take up the microparticles. Microparticles are capable of inducing DC maturation as determined by up-regulation of CD80 and CD86 markers. Although the presence of CpG ODN in the microparticles did not impact on the phenotype of the DCs, it was necessary for DCs to induce activation of antigen-specific T cells as indicated by interferon-gamma production. Microparticles entrapping both antigen and CpG ODN induced significantly higher amounts of anti-OVA antibody production than other preparations such as the soluble OVA and CpG ODN (P<0.01) and stimulated stronger IgG2a production than delivery of microparticles entrapping antigen alone. We conclude that co-encapsulating immunostimulatory CpG ODN and antigen in degradable microparticles is an effective approach to enhancing development of a TH1 immune response.

The remarkable infectiousness of Francisella tularensis suggests that the bacterium efficiently evades innate immune responses that typically protect the host during its continuous exposure to environmental and commensal microbes. In our studies of the innate immune response to F. tularensis, we have observed that, unlike the live vaccine strain (LVS) of F. tularensis subsp. holarctica, F. tularensis subsp. novicida U112 opsonized in pooled human serum activated the NADPH oxidase when incubated with human neutrophils. Given previous observations that F. tularensis fixes relatively small quantities of complement component C3 during incubation in human serum and the importance of C3 to neutrophil phagocytosis, we hypothesized that F. tularensis subsp. novicida may fix C3 in human serum more readily than would LVS. We now report that F. tularensis subsp. novicida fixed approximately six-fold more C3 than did LVS when incubated in 50% pooled human serum and that this complement opsonization was antibody-mediated. Furthermore, antibody-mediated C3 deposition enhanced bacterial uptake and was indispensable for the neutrophil oxidative response to F. tularensis subsp. novicida. Taken together, our results reveal important differences between these two strains of F. tularensis and may, in part, explain the low virulence of F. tularensis subsp. novicida for humans.

We demonstrate spatial control over cell attachment on biodegradable surfaces by flowing cell adhesive poly (D-lysine) (PDL) in a trifluoroethanol (TFE)-water mixture through microfluidic channels placed on a biodegradable poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) substrate. The partial solvent mixture swells the PLA-PEG within the confines of the microfluidic channels allowing PDL to diffuse on to the surface gel layer. When excess water is flowed through the channels substituting the TFE-water mixture, the swollen PLA surface collapses, entrapping PDL polymer. Results using preosteoblast human palatal mesenchymal cells (HEPM) indicate that this new procedure can be used for facile attachment of cells in localized regions. The PEG component of the PLA-PEG copolymer prevents cells from binding to the nonpatterned regions.

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