Potential Salivary Biomarkers In Mastocytosis: A Proteomics Approach

Mastocytosis is a myeloproliferative neoplasm characterized by infiltration of clonally derived mast cells in different tissues. According to mast cells localization, it is possible to discriminate cutaneous mastocytosis (CM) from systemic mastocytosis (SM), the latter involving at least an extracutaneous organ, like bone marrow, liver, spleen and gastrointestinal tract. Some of the SM patient can develop also cutaneous lesions (SM+C). Oral cavity is commonly involved in the symptomatology. Disease classification is often tricky. In the first part of this thesis, in order to highlight possible qualitative/quantitative modifications of the salivary proteome associated to the different forms of the disease, we investigated salivary samples collected from 6 CM, 35 SM patients, among which 8 with only systemic symptoms (SM-C) and 27 with both systemic and cutaneous symptoms (SM+C), and 48 healthy controls by a top-down proteomic approach. Low-resolution HPLC-ESI-MS analysis of the acid soluble fractions of saliva highlighted different proteomic profiles in the three patients’ groups, showing that the salivary samples of the patients were characterized by a down-regulation of peptides and proteins involved in the homeostasis and defense of the oral cavity, and in the innate immunity and in inflammation not only in the oral cavity but at systemic level, such as aPRPs, statherin, histatins and cystatins. Only two proteins with regulatory roles in the innate immunity and inflammation, S100A8 and antileukoproteinase, resulted up-regulated in patients differently to all the other salivary proteins analyzed, suggesting the establishment of a response by the organism to the injuries caused by the disease. Interestingly, some differences have been found among the patients in the concentration of α-defensins 1, thymosin β-4, and the truncated forms of cystatin D-R26 variant, and some truncated forms of P-B and statherin. Correlation between the protein/peptide levels and tryptase concentration evidenced that acidic PRPs, statherin and P-B fragments, and cystatin D-R26 des1-5 correlated positively just in SM-C group, while thymosin β-4 correlated negatively. Since the interesting data on cystatin D, in the second part of the thesis I focused on the characterization of the salivary protein complex aggregating to the cystatin D-C26 variant (named by us SIC-D). Indeed, the C-26 variant is usually undetectable in acid soluble fraction of saliva but measurable in whole saliva. Pools of whole saliva from 4 CM, 3 SM-C, 14 SM+C, and 20 sex/age matched healthy controls, were submitted to immunoprecipitation with cystatin D-C26 antibody followed by SDS-PAGE/western-blot under reducing and non-reducing conditions. Since the low volume of CM samples, the tryptic digestion, and the nano-HPLC-high-resolution-MS/MS analysis were performed only in SM-C, SM+C and control samples. The quantitative comparison was performed with Proteome Discoverer 2.2 software. SIC-D included 44 proteins, among which IgA, IgG, PIgR, annexins, α-defensin 1/2, S100A8, carbonic anhydrase 6, prolactin-inducible protein, lysozyme C and dermicidin. Several qualitative/quantitative differences were highlighted with respect to controls and between the two patient groups. The most relevant were: all the patients exhibited lower levels of IgA, PIgR, DMBT-1 and S100A8 than controls, but higher levels of IgG, α-defensins 1/2 and carbonic anhydrase 6. The highest level of cystatin D-C26 was found in SM+C patients, which were different from SM-C for annexin A2. Both SM-C and SM+C showed the presence of antileukoproteinase and S100A14. The results on the acid-soluble fraction of saliva and the preliminary results on the SIC-D complex are promising in order to find candidate markers able to discriminate the different forms of mastocytosis.