Article Figures & Data
Figures
- Fig. 1.
Growth curves of F. tularensis LVS in the skin, lymph nodes, livers, and spleens of immune (diamonds) and naive (open circles) mice. Mice received a sublethal intradermal inoculum, 3.8 × 105 CFU, of F. tularensis LVS, and bacterial numbers were determined over 15 days. Immune mice had received a sublethal intradermal inoculum 5 weeks before secondary challenge. The line represents the detection limit of the assay. The means for five mice per group and time point are shown.
- Fig. 2.
Expression of TNF-α in skin of infected mice. Samples are from naive (A) and immune (B) mice. For both types of samples, stained section from noninfected tissue (a), 24 h after inoculation (b), 48 h postchallenge (c), and 72 h postinoculation (d) are shown. Controls included staining with irrelevant primary antibodies and the absence of cross-reactivity of the secondary labeled antibodies with the primary antibodies of mismatched isotypes.
- Fig. 3.
Expression of IFN-γ in skin of infected mice. Samples are from naive (A) and immune (B) mice. For both types of samples, stained section from noninfected tissue (a), 24 h after inoculation (b), 48 h postchallenge (c) and 72 h postinoculation (d) are shown. Controls included staining with irrelevant primary antibodies and the absence of cross-reactivity of the secondary labeled antibodies with the primary antibodies of mismatched isotypes.
- Fig. 4.
Expression of IL-12 in skin of infected mice. Samples are from naive (A) and immune (B) mice. For both samples, stained section from noninfected tissue (a), 24 h after inoculation (b), 48 h postchallenge (c), and 72 h postinoculation (d) are shown. Controls included staining with irrelevant primary antibodies and the absence of cross-reactivity of the secondary labeled antibodies with the primary antibodies of mismatched isotypes.
- Fig. 5.
PCR-assisted amplification of cytokine cDNA from skin samples after challenge with F. tularensis LVS. The number of cycles used for amplification was such that constitutive expression in skin was barely discernible or not discernible at all. Samples were taken at indicated time points after intradermal challenge of 5 × 105 CFU of F. tularensis LVS.
- Fig. 6.
Competitive PCR analysis of TNF-α and IFN-γ cDNA levels in skin samples taken at 24 and 48 h postchallenge. The concentration of cDNA was determined by identifying the dilution of the competitor fragment showing the same intensity after amplification as that of the amplicon of the sample cDNA. For each cytokine, the competitor fragment yielded a larger fragment (indicated with an arrow). The IFN-γ competitor DNA ranged, in 10-fold dilutions, from 10−19 to 10−23 mol, and the TNF-α DNA ranged from 10−18 to 10−22 mol.
Tables
- Table 1.
Longevity of immunity memory after immunization withF. tularensis LVS
Organ Mean ± SD CFU of F. tularensisLVS (log10) per sample at indicated time between rechallenge and immunizationa None 1 mo 3 mo Skin 7.8 ± 0.1 2.3 ± 0.1 3.2 ± 0.5 Spleen 5.8 ± 1.1 BDL BDL ↵a The inoculum of the rechallenge was 5 × 105 CFU of F. tularensis LVS administered intradermally. The two groups were reinfected at the same time. Viable counts were determined 3 days after rechallenge for a group of four female, aged-matched mice. BDL, below detection limit (10 organisms/spleen).
- Table 2.
Immunohistochemical analyses of TNF-α, IFN-γ, and IL-12 expression in skin of mice infected withF. tularensis LVSa
Time postchallenge Immunohistochemical staining forb: TNF-α IFN-γ IL-12 Noninfected Naive Immune Noninfected Naive Immune Noninfected Naive Immune None 0.0, 0.0 0.2, 0.0 0.1, 0.4 24 h 0.0, 0.0 0.8, 0.4 0.3, 0.2 0.4, 0.2 0.3, 0.0 2.4, 2.1 48 h 0.2, 0.0 0.6, 0.4 0.1, 0.1 0.4, 0.3 0.2, 0.7 1.3, 0.8 72 h 0.0, 0.5 0.4, 0.0 0.3, 0.6 2.4, 0.8 0.1, 0.5 0.1, 0.2 ↵a Mice were challenged with an intradermal inoculation of saline (none) or with 3.8 × 105 F. tularensis LVS as a primary inoculum (naive mice) or secondary inoculum (immune mice) and killed after 1, 2, or 3 days of infection. One square centimeter of the skin at the site of inoculation was excised and snap frozen. Mice that received a secondary challenge had been given a subcutaneous inoculation of 104 CFU 5 weeks earlier.
↵b Cytokine expression in infectious foci was determined by immunoperoxidase labeling of cryostat sections of biopsy samples with anticytokine antibodies. The number of peroxidase-stained cells in epidermis for each tissue section was determined by scoring the number of positive cells. The scoring system was as follows: 1+ if 1 to 5 cells were stained in each tissue section, 2+ for 5 to 10 cells, and 3+ for >10 cells. The average score for 15 to 30 tissue sections for each organ was calculated. Scores for two mice are given.
