Chronic tonsillar hypertrophy (CTH) is an idiopathic, obstructive lymphoproliferative disease, probably due to continuous antigenic stimulation of an unknown source. The hypothesis to be tested is that CTH is caused by bacterial antigens from metacolonies. This hypothesis is based on our preliminary data: i. Review of limited samples of archival materials found 3 types of bacterial metacolonies, in total, in 80% of CTH cases but only in 7% of non-inflammatory tonsillar biopsies;ii. Metacolonies (n=3) are highly organized polybacterial communities consisting of ~100 species. Members of three genera are possible metacolonycommon species (MC-species);iii. CTH tonsils contain high levels of antibodies to the putative MC-species. We will test the hypothesis by the following 3 Specific Aims.
Specific Aim 1 is designed to determine whether there is a significant correlation between the presence of metacolonies and CTH. We will determine the true occurrence rate of each type of metacolony and inflammatory reaction to them, in adequately sampled CTH tonsils and controls.
Specific Aim 2 is designed to examine whether there are MC-species among metacolonies. Besides cultivation, metacolonies with unique banding patterns on Denaturing Gradient Gel Electrophoresis will be selected for analysis by 16S PCR-based sequencing and in situ examinations to identify putative MC-species. Their conservation in the remaining metacolonies will be validated by species-specific, quantitative real time PCR.
Specific Aim 3 is designed to determine whether B cell proliferation in CTH is a response to antigens from MC-species. Initially, CD19+ B cells will be pulled out by labelling with antigens of the MC-species and single cell sorting, and tested for their ability to produce antibodies to the antigens. Later, we will develop a simple ELISA capable of screening a large number of patients for tonsillar antibodies to the MC-species. Addressing the three aims will provide evidence to examine the hypothesis of a bacterial etiology of CTH.
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