4)

4). The use of mAb complexed to iFt to target iFt to FcR was chosen due to the lack of an identified protective Ag for (28, 31), as well as to a general lack of knowledge regarding which specific FcR might be involved in immune responses at mucosal sites. be lethal when inhaled (28). Ergoloid Mesylates Both cellular and humoral immunity have now been shown to mediate varying levels of protection against infection with this organism, but no approved vaccine is currently available (28C33). Therefore, these studies are designed to determine whether iFt, when targeted to FcRs in the form of mAb-iFt at a mucosal site, would enhance protection against challenge with this infectious agent. Importantly, results of Ergoloid Mesylates these studies fill a significant gap in our knowledge regarding the use of FcR-targeted Ag as an immunogen, while establishing FcR targeting as an alternative and viable vaccine strategy for enhancing mucosal immunity to both intracellular and extracellular pathogens. Materials and Methods Mice BALB/c and C57BL/6 mice were procured from Taconic Farms. IgA?/? mice with a C57BL/6 129 background were bred at Albany Medical College (Albany, NY). The B6.1291-Fcrgttm1Dcr/Dcr (FcRn?/?) and (C57BL6 129) F1 hybrids were obtained from The Jackson Laboratory, while FcR?/? mice on C57BL/6 129 background were procured from Taconic Farms. All mice were housed in the Animal Resources Facility at Albany Medical College. Mice were provided with ad lib water and food during the course of each experiment. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee. Cells RAW 264.7 cells are a FcRI/FcRIIB-expressing mouse myeloid cell line (American Type Culture Collection). Cells were grown in DMEM with 4.5 g/L glucose and L-glutamine without sodium pyruvate (Mediatech) containing 10% FBS (HyClone) and 1 mg/ml gentamicin (Life Technologies). Generation of immunogen (iFt and mAb-iFt complexes) GFP-expressing LVS organisms were provided by Dr. Mats Forsman (Swedish Defense Research Agency, Stockholm). GFP-expressing organisms were used to facilitate the monitoring of iFt vs mAb-iFt binding to myeloid FcRs. To inactivate these organisms, 1 1010 CFU/ml live GFP-expressing LVS were incubated in 1 ml of sterile PBS (Mediatech) containing 2% paraformaldehyde (Sigma-Aldrich) for 2 h at Ergoloid Mesylates room temperature. Fixed bacteria (iFt) were then washed with sterile PBS three times. Importantly, while very resistant to fixation, GFP fluorescence may be reduced as a result (34). However, as indicated in Ergoloid Mesylates subsequent studies, GFP fluorescence was maintained despite fixation. Furthermore, any reduction in fluorescence is most likely uniformly distributed, and the ability to detect differential binding of iFt vs mAb-iFt to myeloid cells was not affected. Inactivation was verified by culturing a 100-l sample (1 109 CFU) of the iFt on chocolate agar plates (Fisher Tm6sf1 Scientific) for 10 days. To make mAb-iFt, mouse IgG2a anti-LPS mAb (Fitzgerald Industries International) was added at a final concentration of 1 1, 5, 10, and 30 g/ml to iFt in PBS (1 109 CFU/ml) and then incubated overnight on a rocker at 4C. Following overnight incubation, the mAb-iFt complexes were washed three times with sterile PBS and then resuspended in 1 ml of PBS. Analysis of iFt and mAb-iFt binding to Fc receptors Binding of mAb-iFt to FcR-bearing RAW cells was visualized by flow cytometry. RAW cells were washed three times with PBS/BSA/azide. Next, 5 105 cells in 20 l PBS containing 0.02% azide (Sigma-Aldrich) was added to each well of a 96-well plate, followed by.