Levels of N-PmpC-specific IgG in the sera of BALB/c mice immunized via the conjunctiva or 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. The statistical significance of the observed differences was evaluated using 1-way repeated ANOVA (<i>p <0</i>.<i>05*</i>, <i>p <0</i>.<i>005</i>**, <i>p <0</i>.<i>0001</i>***). In all cases when the non-immunized group was not a reference, compared groups are indicated by arrows.</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

The proliferation indices (PI) of N-PmpC-stimulated splenocytes from BALB/c mice immunized according to the assigned immunization protocol.

<p>The numbers of viable splenocytes were assessed by <i>Cell</i> Counting Kit-<i>8</i> assay following a 48 h cultivation in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with N-PmpC (10 μg/ml). Each dot represents PIs calculated for individual mouse. Lines indicate the mean values [A_492/620 ± SD (n = 10)] calculated for each group. The statistical significance of the observed differences in PIs between groups treated according to the assigned protocols was evaluated using 1-way repeated ANOVA. <i>(p < 0</i>.<i>05*</i>, <i>p < 0</i>.<i>005**</i>, <i>p <0</i>.<i>0001***)</i>. Non-immunized group was used as a reference unless otherwise indicated by two head-arrow.</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 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

Ocular conjunctiva of N-PmpC EcN BGs-treated and control guinea pig eyes.

<p>Guinea pigs were exposed to N-PmpC EcN BGs two and 24 h. Conjunctival epithelia from both N-PmpC EcN BGs-treated and control displayed normal cell layers and morphology. No sign of tissue edema were observed in the conjunctiva studied after exposure to N-PmpC EcN BGs compared with controls. Scale bar: 10 μm. Magnification 40x.</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

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