Salivary histatins degradation in Down syndrome subjects and antibacterial influence on periodontopathogens

Abstract

The aim of this study is to evaluate the proteolysis of histatins 1 and 5 in whole saliva of subjects with Down syndrome (DS) and periodontal disease (PD), as well the antibacterial function of the degradation products. Twenty four subjects will be selected and equally divided in the following groups (n=6): DS with PD (Group DS/+PD), DS without PD (Group DS/-PD), non-syndromic with PD (Group NS/+PD) and non-syndromic without PD (NS/-PD - control group). First, periodontal condition, DMFT index and ICDAS will be evaluated and stimulated whole saliva will be collected to obtain 3.0-4.0 ml. Then, saliva will be aliquoted and stored in -80°C, being part centrifuged in order to obtain the supernatant of whole saliva (WSS), and the not centrifuged samples will be used in microbiological analysis. Levels of Porphyromonas gingivalis e Aggregatibacter actinomycetemcomitans will be quantify by Real-time Polymerase Chain Reaction (qPCR). Protein degradation will be performed by adding synthetic histatins 1 or 5 to pure WSS and diluted SST (1:10). The samples will be incubated at 37°C for 0, 0.5, 1.5, 4, 6, 8, 24 e 48 hours. Next, reverse-phase HPLC, polyacrylamide cationic gel electrophoresis (PAGE) and mass spectrometry will be performed. To test bacterial growth inhibition, histatins 1 or 5 will be diluted in BHI supplemented broth and strains of Porphyromonas gingivalis (ATCC 33277) and Agreggatibacter actinomycetemcomitans (JP2) will be added to this solution in concentrations of 1x104 CFU/mL. Plates will be incubated for 48 hr at 30ºC in anaerobic jar and growth will be measured by optical density at 620 nm. The values will be corrected by absorption of BHI and the values of inhibitory concentration (IC50) will be determined by growth inhibition curve. Statistical tests will be defined based on variation factors of the experiments and after data analysis as its distribution and homoscedasticity. The significance level will be 5%.