Calculo de mediciones de la altura de árbol

surface at 24 h and 48 h are shown in Table 13. The density of bacteria adhering to each mucosal feature has been calculated by dividing the total number of bacteria by the percent of the mucosal surface occupied by each feature and

Figure 17

Cell damage with cell debris in an organ culture infected by PL+ at 24 h.

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Figure 18

Organ culture infected with Streptococcus pneumoniae PL+ for 24 h. The cilia of PL+ infected organ cultures appear disorganised and bent in different directions compared to those in control organ cultures.

Figure 19

Organ culture infected with Streptococcus pneumoniae PL+ for 24 h showing extrusion of ciliated cells.

Table 12

Scanning electron microscopy of the interaction between Streptococcus pneumoniae and adenoid organ culture

Strain Mucus Damaged

mucosa Ciliated epithelium Unciliated epithelium 4 h CONTROL PL+ PL- 48.6±7.6 32.3±16.7 33.0±17.6 4.3±0.9 8.4±2.9 6.1±2.8 22.2±16.0 21.2±10.5 25.4±18.8 24.7±11.7 38.1±16.9 35.5±7.4 24 h CONTROL PL+ PL- 48.4±20.6 31.4±15.5 41.8±21.2 4.3±1.4 21.7±6.8*+ 8.8±2.8 25.1±9.1 8.0±6.5* 16.2±13.2 2 2 .2±12.5 38.9±11.8 3 3 .2±19.6 48 h CONTROL PL+ PL- 39.5±22.3 45.4± 7.8 36.0±19.2 5.0±2.6 30.0±4.7** 23.4±10.5+ 1 9 .5±6 .2 4.9±2.7* 7.5±5.7+ 3 6 .0±12 .3 1 9 .7±2.!♦ 33.1±17.0 *p<0.01, PL+ cf control + p<0.02, PL+ cf PL- **p<0.005, PL+ cf control ♦p<0.05, PL- cf control; PL+ cf control

The results are the mean percentage surface area of an organ culture occupied by each mucosal feature ± standard deviation, At each time point an experiment (n=6) consisted of three organ cultures constructed from the same adenoid tissue. PL+ and PL- are pneumolysin sufficient and deficient S.pneumoniae

a) Mucus

At 24 h PL+ and PL- were most commonly found in association with mucus, and there was no significant difference in adherence to mucus for the two variants (p<0.7). PL+ adherence to mucus was significantly increased compared to ciliated and unciliated tissue (p<0.05). In infected cultures the mucus became fibrogranular and appeared to contain both cellular material and bacteria, which was not seen in control cultures (Figure 20) . By 48 h the two variants again demonstrated tropism for mucus compared to ciliated and unciliated tissue (Tables 13 and 14). Projections from bacteria were seen (Figure 21).

b) Damaged cells

At 24 h PL+ and PL- were seen to adhere to damaged cells. Although the total number of PL+ adhering to areas of damage was greater than P L - . When the bacterial density was measured there was no difference suggesting that there was no difference in adherence of PL+ and PL- to damaged cells (Figure 22) . Bacteria also adhered to damaged ciliated cells which appeared to be undergoing extrusion

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Figure 2 0

Organ culture infected by Streptococcus pneumoniae PL+ for 24 h. The mucus appears fibrogranular and appears to contain both cellular material and bacteria.

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Figure 21

Organ culture infected by Steptococcus pneumoniae PL+ for 48 h. The mucus appears fibrogranular and projections from bacteria are seen.

c) Cilia

PL+ were only rarely seen in association with cilia at 24 h and 48 h. PL- bacteria were not seen in association with cilia at 24 h but were occasionally seen at 48 h.

d) Unciliated epithelium

Adherence to unciliated epithelium was uncommon for both variants, although the total number of PL+ adhering to unciliated epithelium was greater than PL- (p<0.05). The reason for this was PL+ adherence to normal unciliated cells at sites where separation of tight junctions had occurred. This separation of cells, that otherwise appeared normal, was seen exclusively in PL+ infected cultures.

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Figure 22

Organ culture infected by Streptococcus pneumoniae PL+ for 48 h. PL+ bacteria were seen adhering to both areas of cell damage and to areas of unciliated epithelium where a separation of tight junctions integrity could be seen.

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Figure 2 3

Organ culture infected by Streptococcus pneumoniae PL+ for 48 h. PL+ adherence to damaged ciliated cells which appear to be undergoing extrusion.

Table 13

Bacterial adherence to the mucosal surface of adenoid organ culture assessed by scanning electron microscopy

Mucus Damaged mucosa Ciliated epithelium Unciliated epithelium 24 h PL+ 19.2±8.9 9.8*± 4.5 1.1±1.6 6.2*±2.7 PL- 16.7±9.9 4.8 ± 3.0 0 2.3 ±2.3 48 h PL+ 142.0±14.7 121.7*± 8.4 2.7±2.4 23.8*±8.7 PL- 128.7±23.6 100.1 ±26.6 1.0±2.7 14.2 ±8.5 * = p<0.05, PL+ cf PL-

The results are the mean number of bacteria adhering to each mucosal feature ± standard deviation. At each time point an experiment (n=6) consisted of three organ cultures (including control) constructed from the same adenoid tissue. PL+ and PL- are pneumolysin sufficient and deficient variants respectively

Table 14

Bacterial density on each mucosal feature of adenoid organ culture Mucus Damaged mucosa Ciliated epithelium Unciliated epithelium 24 h PL+ 3 .7±1.0* 2.0±1.8* 0.8±1.7 0.43±0.27 PL- 2 .3±2.2 1.5±1.4 0 0.16±0.19 48 h PL+ 7.7±2.0t 10.3± 1.5t 1.3±1.0 3.0 ± 1.2 PL- 8.6±6.04 8.9 ±0.7 0.4±0.8 1.7 ± 1.4

* PL+ adherence to mucus and damaged cells compared to ciliated and unciliated tissue p<0.05

t PL+ adherence to mucus and damaged cells compared to ciliated and unciliated tissue p<0.02

♦ P I - adherence to mucus and damaged cells compared to ciliated and unciliated tissue p<0.05

The results are the mean (n=6) total number of bacteria adhering to each mucosal feature divided by the percent of the mucosal surface occupied by that feature ± standard deviation. At each time point an experiment consisted of three organ cultures

(including control) constructed from the same adenoid tissue. PL+ and PL- are pneumolysin sufficient and deficient variants