Bacterial load and IFN expression during the course of primary infection.

<p><b>A.</b> Course of primary infection in mice with the wild type <i>Lm</i> (EGD-e) and the recombinant <i>L. inn</i>::vgc strain. Mice were infected i.v. with 10³ cfu <i>Lm</i>, 10⁷ cfu <i>L.inn</i>, or 10⁷ cfu <i>L.inn::vgc</i> strains. At different time intervals after the infection, mice were sacrificed and the number of viable bacteria in the organs was enumerated. <b>B.</b> Quantitative measurement of IFNα2 and IFNb1 expression in bone marrow-derived macrophages using RT-PCR at 2 h and 8 h following infection with <i>Lm</i> , <i>L.inn</i>, or the <i>L.inn</i>::<i>vgc</i> strains. *P<0.05 (<i>L.inn</i> vs. <i>Lm</i> and <i>L.inn::vgc</i> strains).</p

Measurement of proinflammatory cytokine levels in serum.

<p>Sera was obtained from mice on days 1, 2, 3, and 4 post-infection after inoculation with 10³ cfu <i>Lm</i>, 10⁷ cfu <i>L.inn</i>, or 10⁷ cfu <i>L.inn::vgc</i>. Levels of IL-1ß, IL-6, IL-12(p70), and TNF-alpha were quantified using a multiplex cytokine assay kit. *P<0.05 (EGD-e vs. <i>L.inn</i> and <i>L.inn::vgc</i> strains).</p

Examination of spleens and DTH response after infection with <i>Lm</i> and the recombinant <i>L.inn::vgc</i> strain.

<p><b>A.</b> Morphological examination of spleens from mice inoculated i.v. with the wild type <i>Lm</i> and the recombinant <i>L.inn::vgc</i> strain. Spleens of mice infected i.v. as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035503#pone-0035503-g001" target="_blank">Fig. 1</a> were isolated on day 3 after infection. Shown is a spleen from mice infected with the wild type <i>Lm</i>, the wild type <i>L.inn</i> and its recombinant mutant strain <i>L.inn::vgc</i>. Infiltration of monocytic cells and granulomatous lesions are only detectable in the spleens isolated from mice infected with the wild type <i>Lm</i>. <b>B.</b> Spleen sections were stained with HE and examined. Granulomas with massive leukocyte aggregates can only be detected in spleens of mice infected with <i>Lm</i>. <b>C.</b> DTH response to listerial antigen 9 days after primary infection. Mice were infected with 10³ CFU of <i>Lm</i>, 10⁷ CFU of <i>L.inn</i>, or 10⁷ CFU of <i>L.inn::vgc</i> strain. 9 days after infection, DTH was triggered through injection of soluble somatic listerial antigen. Twenty-four hours later, the specific skin response was determined. The mean value ± S.E. of five animals of a representative experiment is shown.*P<0.05 (EGD-e vs. <i>L.inn::vgc</i> strain).</p

Protective immunity and cellular immune response after infection with <i>Lm</i> and the <i>L.inn::vgc</i> strain.

<p><b>A.</b> Induction of protective immunity conferred after infection with the <i>L.inn::vgc strain</i>. Groups of 15 mice were infected i.v. as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035503#pone-0035503-g001" target="_blank">Fig. 1</a>. Two months later all mice were challenged with a lethal dose (20×LD₅₀) of the wild type <i>Lm</i>. As a control, a group of uninfected normal mice was included. Survival of mice after the challenge was monitored up to 8 days. <b>B.</b> Number of antigen-specific IFN-gamma producing CD8+ T cells in spleens of mice infected i.v. with the wild type <i>Lm</i>, <i>L.inn</i> and <i>L.inn::vgc</i> strain determined by ELISPOT. Spleen cells from infected mice were isolated either on day 9 after the primary infection or day 5 after challenge infection and stimulated with the immunodominant MHC class I peptide LLO_91–99 in triplicates in nitrocellulose based 96-well culture plates. Number of specific IFN-gamma producing cells against the dominant H-2K^d restricted LLO_91–99 epitope were determined by counting the number of spots under the microscope. *P<0.05 (<i>L.inn</i> vs. <i>Lm</i> and <i>L.inn::vgc</i> strains).</p

Expression levels of CD62L on CD8⁺ splenocytes following primary and recall infection with <i>Lm</i>, <i>L.inn</i> and the <i>L.inn::vgc</i> strain.

<p>Flow cytometry was performed on spleen cells, isolated from mice on day 60 after the primary infection or day 5 after the challenge. Cells were stained with FITC-labelled anti-Lyt-2 and biotinylated anti-CD62L. The binding of anti-CD62L on the cell surface was detected with PE-conjugated streptavidin. Numbers shown are gated CD8⁺CD62^lo T cells and analyzed with CELLQuest software.</p

RNA expression pattern of the <i>bcl-2</i> family genes in the testis.

<p>The expression of the anti-apoptotic gene <i>bcl-2</i> (<b>A</b>), pro-apoptotic genes <i>bax</i> (<b>B</b>), <i>bid</i> (<b>C</b>), <i>bim</i> (<b>D</b>) and <i>bak</i> (<b>E</b>) in the testis were determined with quantitative real time PCR. Target gene expression levels were normalized with the endogenous control ß-2-microglobulin (ß2M). Data are present as 2^ΔCt, ΔCt = Ct_{target gene}-Ct_ß2M. The Mann-Whitney U test was employed for statistical analysis (* <i>p</i><0.05). Each single symbol (circle and triangle) represents one individual testis sample.</p

Increase of TUNEL positive cells in UPEC infected testis.

<p>(<b>A</b>) DNA strand breakage in testicular cells from control (upper panel) and UPEC infected (lower panel) rats were analyzed using TUNEL assay. Nuclei were counterstained with DAPI (blue). TUNEL (+) cells (green) with ring-like nuclear stain are indicated with arrows. (<b>B</b>) Numbers of TUNEL (+) cells are presented as mean ± SD/seminiferous tubule. Student’s t-test was used for statistical analysis and the level of significance is indicated as **<i>p</i><0.01. (x20 objective).</p

NF-κB pathway is not activated in UPEC infected testis.

<p>(<b>A</b>) Total testis proteins were separated on 10% SDS-PAGE. Immunoblots were labeled with mouse anti-IkBα antibody. ß-actin served as a loading control. (<b>B</b>) The intensity of target bands was measured with the ImageJ software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) and results are presented as the relative intensity = intensity of p65/intensity of ß-actin. (<b>C</b>) Testis cryosections were probed with anti-p65 antibody labeled with Cy3-linked secondary antibody (orange) and the nuclei were counterstained with DAPI (blue, images taken with x40 objective). Representative results from at least two independent experiments are shown.</p

Electron microscopical analysis reveals intact blood-testis barrier (BTB) and blood-epididymis barrier (BEB).

<p>(<b>A</b>) Ultrastructural analysis shows that intercellular tracer penetration does not extend beyond the junctional complex of the BTB (arrow in inset) within the seminiferous epithelium of UPEC infected rats (x3,000 magnification, inset x20,000 magnification). SC = Sertoli cells, orientation of the luminal and basal compartment are highlighted (<b>B</b>) Ultrastructural analysis of a UPEC infected epididymis demonstrates intercellular tracer penetration (x3,000 magnification). Inset is a magnification of the area represented in the black frame showing the tight junctions. (x20,000 magnification).</p

Caspase-1, -3, -6 and-8 are not activated in UPEC infected testis.

<p>Total testis protein (20 µg) from four different animals in each group were separated on 15% SDS-PAGE. Immunoblots were probed with anti-caspase-8 (<b>A</b>), anti-caspase-3 <b>(B, upper panel)</b>, anti-caspase-6 <b>(B, lower panel)</b> and anti-caspase-1 (<b>C</b>) antibodies and detected using chemiluminescence. RAW 264.7 cells treated with sodium nitroprusside (SNP) served as a positive control. (<b>D</b>) The intensity of target bands on the films was measured with the ImageJ software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). Semi-quantitative results are presented as mean ± SD and Student’s t-test was used for data analysis (Caspase-8, <i>p = </i>0.875; Caspase-3, <i>p = </i>0.686; Caspase-6, <i>p = </i>0.486; Caspase-1, <i>p = </i>0.343).</p