Objectives:
To investigate:
Expression of TLRs in chemotherapy-induced mucositis
The role of oral microflora in TLR expression
Methods:
Non-transformed rat intestinal epithelial cells (IEC-6) were seeded into two 12-well culture plates and incubated until confluent. Transwell® permeable membrane inserts were suspended over each well. Saliva samples (healthy patient) in BHI broth were added to the inserts, and SN-38 (irinotecan active metabolite) was added to the wells, in the following configurations for each plate:
3 wells with saliva sample and SN-38
3 wells with saliva sample, but no SN-38
3 wells with SN-38, but no saliva sample
3 double negative controls
Cells were incubated for 24 or 48hrs (37°C, 5% CO2) prior to harvesting. Samples were prepared and SYBR-Green based RT-PCR testing was performed. Pfaffl’s model for relative quantification was used. Results were subjected to two-way ANOVA and Tukey’s multiple comparison tests.
Results:
At 48hrs, there was significantly greater expression of TLR4 in samples with salivary microbes compared to their microbe-free counterparts, in both the SN-38 negative and positive groups (95% CI, p<0.05). The presence of SN-38 did not result in significantly different TLR4 expression.
At the 24hr timepoint, differential expression of TLR4 between groups did not reach significance.
Conclusions:
Preliminary results suggest that the presence of microbes is more influential in TLR4 expression than irinotecan. Testing of TLRs 2, 5, and 9 is yet to be finalised.