the trachea. Tissue was fixed in formalin, embedded in paraffin, and sections were stained with standard hematoxylin and eosin (H&E). Lymphocytic infiltration was analyzed by researchers ‘blinded’ to sample identity, and morphometry was determined using a tool of an Axioplan microscope (Zeiss). The lymphoid areas were defined by the squared micron value for each cluster of lymphocytes encountered in the lung parenchyma. Total area covered by lymphoid follicles was calculated by adding the individual areas occupied by all the lymphoid follicles present in 2 the whole lung section and expressed in squared microns (mm ). Immunofluorescence and morphometric analysis of BALT Formation of germinal centers, B cell interaction with T cells, and B cell proliferation were evaluated with a combination of commercial antibodies. B cells were detected with biotinylated antibodies against B220 (clone: RA3-6B2; BD Pharmingen, San Diego, CA) followed by the addition of Streptavidin-Alexa Fluor 488 (Molecular probes, Eugene, OR) or Streptavidin-Alexa fluor 594 (Molecular probes, Eugene, OR). For detecting T cells (CD3- epsilon, clone: M-20; Santa Cruz Biotechnology, Santa Cruz, CA) and proliferating cell nuclear antigen (PCNA, clone C-20; Santa Cruz Biotechnology, Santa Cruz, CA) slides were first incubated with purified goat-anti mouse CD3 and goat anti PCNA. In a second step, CD3 and PCNA were visualized with donkey anti- goat, conjugated to Alexa Fluor 594 (Molecular probes, Eugene, OR). Germinal center B cells were detected by incubating slides with peanut agglutinin-FITC (PNA-FITC) (SIGMA, St. Louis, MO), followed by rabbit anti-FITC, conjugated to Alexa Fluor 488 (Molecular probes, Eugene, OR). Dendritic cells were detected with a combination of hamster-anti mouse CD11c-PE (clone HL3, BD Pharmingen, San Diego, CA) and rat-biotin anti mouse CD11b (M1/70, BD Pharmingen, San Diego, CA), followed by incubation with rabbit anti-PE (Rockland Immuno- chemicals for Research Inc, Gilbertsville, PA) and Streptavidin FITC (BD Pharmingen, San Diego, CA) and finally PE was detected with anti-rabbit Cy-3 (Jackson Immunoresearch, West Grove, PA). For detecting immunoglobulin producing cells, lung sections were stained with a combination of antibodies directed against goat anti PCNA (Santa Cruz Biotechnology, clone C-20), biotin, rat anti IgM (BD Pharmingen), rat anti-IgA (BD Pharmingen, clone C-10) or FITC-donkey anti-mouse IgG (Jackson Immunoresearch). For T cells we used goat anti CD3 (Santa Cruz Biotechnology, clone M20), and we combined it with anti-PCNA antibody for the identification of proliferating T cells. Anti-PCNA specifically stains nuclei (red), while anti-CD3 stains References 1. Di Tommaso A, Saletti G, Pizza MG, Rappuoli R, Dougan G (1996) Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant. Infect Immun 64: 974–979. 2. Hirabayashi Y, Kurata H, Funato H, Nagamine T, Aizawa C, et al. (1990) Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine 8: 243–248. 3. Ciabattini A, Giomarelli B, Parigi R, Chiavolini D, Pettini E, et al. (2008) Intranasal immunization of mice with recombinant Streptococcus gordonii expressing NadA of Neisseria meningitidis induces systemic bactericidal antibodies and local IgA. Vaccine 26: 4244–4250. 4. Wu HY, Russel MW (1993) Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin subunit B. Infect Immun 61: 314–322. 5. Neutra RM, Kozlowski PA (2006) Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 6: 148–158. 6. Chen H (2000) Recent advances in mucosal vaccine development. J Control Release 67: 117–128. 7. Wu TH, Hutt JA, Garrison KA, Berliba LS, Jhou Y, et al. (2005) Intranasal vaccination induces protective immunity against infection with virulent Francisella tularensis biovar A. Infect Immun 73: 2644–2654. Tularemia Vaccination and BALT the cell surface (red), allowing the distinction between the two signals. Goat antibodies were detected with donkey anti-goat, Alexa fluor 568 (Invitrogen) and rat antibodies were detected with donkey anti-rat, Alexa fluor 488 and streptavidin-alexa fluor 488 (Invitrogen). FITC signal was amplified with rabbit anti-FITC, alexa fluor 488. Homeostatic chemokines were detected with goat anti mouse CXCL13 and goat anti mouse CCL21. Follicular dendritic cell (FDC) networks were detected with antibodies against FDCM-1 (BD Pharmingen) and biotin- rat anti mouse CD21-CD35 antibodies. B cells were stained with APC-rat anti CD45R (BD Pharmingen). To visualize chemokines and cells, slides were incubated with donkey anti-goat, conjugated to alexa fluor 568 and with donkey anti-rat, alexa fluor 488 and streptavidin-alexa fluor 488 (Invitrogen). Slow fade gold antifade with DAPI (Molecular probes, Eugene, OR) was used to counterstain tissues and to detect nuclei. Images were obtained with a Zeiss Axioplan 2 microscope and recorded with a Zeiss AxioCam digital camera. Whole lungs from four mice per group underwent morphometric analysis in a blinded manner using the morphometric tool of Zeiss Axioplan microscope (Zeiss), which determines the area defined by the squared pixel value for each B cell follicle. Total area occupied by B cell follicles was calculated as mentioned above. Statistical analysis Differences between experimental groups were determined by the Mann-Whitney U nonparametric test by using GraphPad Prism software version 4.02 (San Diego, CA). P values #0.05 were considered significant, while P values #0.01 were considered highly significant. Acknowledgments We are grateful to Dr Milan Blake for kindly providing the E. coli strain expressing meningococcal PorB, and to Dr Paola Massari for valuable scientific advice regarding the use of this protein and discussion on data analysis. We also thank Drs Adalberto Pessoa Ju´nior and Marco Antoˆnio Stephano for their support of Laura Oliveira-Nascimento during her time spent in Dr Wetzler’s laboratory. Author Contributions Conceived and designed the experiments: DC LW. Performed the experiments: DC JRM GB KC LON SW JA. Analyzed the data: DC JRM TDR LW. Contributed reagents/materials/analysis tools: JRM SW JA TDR LW. Wrote the paper: DC LW. 8. Pammit MA, Raulie EK, Lauriano CM, Klose KE, Arulanandam BP (2006) Intranasal vaccination with a defined attenuated Francisella novicida strain induces gamma interferon-dependent antibody-mediated protection against tularemia. Infect Immun 74: 2063–2071. 9. Bakshi CS, Malik M, Mahawar M, Kirimanjeswara G, Hazlett KRO, et al. (2008) An improved vaccine for prevention of respiratory tularemia caused by Francisella tularensis SchuS4 strain. Vaccine 26: 5276–5288. 10. Pechous RD, McCarthy TR, Mohapatra NP, Soni S, Penoske RM, et al. (2008) A Francisella tularensis Schu S4 Purine auxotroph is highly attenuated in mice but offers limited protection against homologous intranasal challenge. PLoS ONE 3: e2487. 11. Oyston PCF, Quarry JE (2005) Tularemia vaccine: past, present and future. Antonie Leeuwenhoek 87: 277–281. 12. Chiavolini D, Weir S, Murphy JR, Wetzler LM (2008) Neisseria meningitidis PorB, a Toll-Like Receptor 2 Ligand, Improves the Capacity of Francisella tularensis Lipopolysaccharide To Protect Mice against Experimental Tularemia. Clin Vaccine Immunol 15: 1322–1329. 13. Conlan JW, Shen H, Webb A, Perry MB (2002) Mice vaccinated with the O- antigen of Francisella tularensis LVS lipopolysaccharide conjugated to bovine serum albumin develop varying degrees of protective immunity against systemic PLoS ONE | www.plosone.org 12 June 2010 | Volume 5 | Issue 6 | e11156