The proliferation indices (PI) of TTd-stimulated SMLN cells from BALB/c and C57BL/6 mice immunized via the conjunctiva according to the assigned immunization protocol.

<p>The numbers of viable SMLN cells were assessed by MTT assay following a 48 h cultivation in 10% FCS/50 µM β-mercaptoethanol/RPMI 1640 medium supplemented or not with TTd (5 µg/ml). PIs were calculated for each mouse. The results are presented as the mean PI ± SE for each experimental group (n = 10). The statistical significance of the differences in PIs between groups treated according to the assigned protocols was determined by <i>t</i>-test (<i>P<</i>0.05*, <i>P<</i>0.005**). The reference group is indicated by a dotted line, and the comparison group is indicated by an arrow.</p

The relative abundance (RA) of TTd-specific mIgG⁺ B cells within the total population of mIgG⁺ B cells in SMLN upon completion of the assigned immunization protocols.

<p>The RA of the TTd-specific mIgG⁺ B cell population was calculated for each mouse. The results are presented as the mean RA ± SE for each experimental group of mice (n = 5). The statistical significance of the observed differences in TTd-specific mIgG⁺ B cell pool abundances was determined by <i>t</i>-test (<i>P<0.05*</i>, <i>P<0.005**</i>, <i>P<0.0005***</i>). The reference group is indicated by a dotted line, and an arrow indicates the comparison group.</p

Levels of IFNγ (A), IL-4 (B), IL-17A (C) and IL-10 (D) in the supernatants from <i>in vitro</i> TTd-stimulated SMLN cells obtained from age-matched control mice (n.c.) and mice immunized via the conjunctiva according to the assigned protocol (bars).

<p>SMLN cells were cultivated at 37°C in 5% CO₂ for 48 h in 10% FCS/RPMI 1640/50 µM β-mercaptoethanol supplemented with 5 µg/ml TTd. The levels of cytokines in the supernatants of corresponding SMLN cells incubated in 10% FCS/RPMI 1640/50 µM β-mercaptoethanol under similar conditions (non-stimulated cells) are indicated by solid lines. The results are presented as the mean concentration ± SE (n = 10). Concentrations of cytokines in supernatants of TTd-stimulated cultures were compared by <i>t-</i>tests. The reference group is indicated by a dotted line, and the comparison group is indicated by an arrow. A <i>t</i>-test was also used for the comparison of cytokine concentration in supernatants of corresponding non-stimulated and TTd-stimulated cultures, and the statistical significance of the differences is marked next to the solid bar, indicating the level of the cytokine within the non-stimulated culture. The levels of statistical significance are assigned as follows: <i>P<0.05*</i>, <i>P<0.005**</i>, <i>P<0.0005***</i>_.</p

Levels of TTd-specific SIgA in tear washes from BALB/c and C57BL/6 mice that were immunized according to the assigned protocols.

<p>Samples were collected two weeks after the completion of the immunizations and were assayed by ELISA (dilution 1∶2). The results are presented as the mean A_492/620± SE (n = 10). The significance of the observed differences was calculated by <i>t</i>-test (<i>P<0.05*</i>, <i>P<0.005**</i>). The reference group is indicated by a dotted line, and the comparison group is indicated by an arrow.</p

IgG1/IgG2a (A) and IgG1/IgG2c (B) ratios calculated for TTd-specific antibodies in the sera of TTd-immunized BALB/c and C57BL/6 mice, respectively.

<p>Serum samples were collected two weeks after the completion of the immunizations and were assayed by ELISA (dilution 1∶100). Ratios were determined using the A₄₀₅ values obtained upon the measurement of TTd-specific IgG1 and IgG2a or IgG2c in sera. The results are presented as the mean A₄₀₅(IgG1)/A₄₀₅(IgG2a,c) ± SE (n = 10). The significance of the differences between syngeneic mice immunized subcutaneously with TTd (reference group) and those immunized via the conjunctiva was calculated by <i>t</i>-test (<i>P<</i>0.05*, <i>P<</i>0.005**, <i>P<</i>0.0005***).</p

Levels of N-PmpC-specific IgA in the sera of BALB/c mice immunized via conjunctiva and subcutaneously.

<p>Serum samples were collected two weeks after the completion of the immunizations and were assayed by ELISA (dilution 1:100). Each dot represents one sample. Lines indicate the mean values [A_492/620 ± SD (n = 10)] calculated for each group.</p

Guinea pigs were immunized with N-PmpC EcN BGs and plain EcN BGs via the conjunctiva and subcutaneously, on days 0, 14 and 28 (50 μg BGs per dose) and two weeks after the completion of the specified immunization protocol were challenged by the ocular administration of 1 x 10⁶ IFU/animal.

<p>The animals were visually assessed and scored on a daily basis. Results are presented as mean pathology score ± SD at defined time point for all animals within a group (n = 5 per group).</p

Levels of anti-N-PmpC mucosal IgA in tear washes from BALB/c mice obtained by two routes of immunization.

<p>Samples were collected two weeks after the completion of the immunization schedule and were assayed by ELISA (dilution 1:10). Each dot represents one sample. Lines indicate the mean values [A_492/620 ± SD (n = 10)] calculated for each group. The statistical significance of the observed differences was evaluated using 1-way repeated ANOVA using non-immunized group as a reference (<i>p <0</i>.<i>05*</i>, <i>p <0</i>.<i>005</i>**, <i>p <0</i>.<i>0001***</i>).</p

Levels of IFNγ (A) and IL-4 (B) in the supernatants from splenic cultures of N-PmpC EcN BGs-, EcN BGs- and non-immunized BALB/c mice.

<p>Mice were immunized subcutaneously (s.c.//) or via the conjunctiva (conj//). Each group consisted of 10 mice. Splenocytes were cultivated at 37°C in 5% CO₂ for 48 h in 10% FCS/RPMI 1640/50 μM β-mercaptoethanol supplemented with 10 μg/ml N-PmpC (+) or without any additional supplementation (-). Differences in concentrations of cytokines in culture supernatants were evaluated using a 1-way repeated ANOVA (<i>p <0</i>.<i>05*</i>, p <i><0</i>.<i>005**</i>_,<i>p < 0</i>.<i>0001***</i>). Corresponding (non-stimulated and N-PmpC-stimulated) non-immunized group cultures were used as a reference unless otherwise indicated by two head-arrow.</p

The expression of the N-PmpC was quantified using Western blot.

<p>BGs expressing major outer membrane protein (MOMP) were used as standards. Line 1, molecular weight markers; line 2, N-PmpC expressed within EcN BGs (MW 92.1 kD), line 3, 560ng MOMP standard 1; line 4, 760ng MOMP standard 2; line 5, 1200ng MOMP standard 3; line 6, 1600ng MOMP standard 4. The membrane was developed using anti-myc-HRP antibody. Quantification of the N-PmpC was performed with the QuantityOne Software in the ChemiDocXRS program.</p