Dysbiosis, i.e. the alteration of the gut microbiota, as well as alterations of the gastrointestinal (GI) barrier, play a crucial role in human health and disease. The interest in elucidating the role of such conditions in diseases is growing, also due to the wide possibility of intervention to heal the gut function. In parallel, there is an increasing demand for effective methods aimed to evaluation of dysbiosis and GI barrier alterations, in a non invasive and cost-effective manner, since this is needed both for baseline patient characterization and for monitoring patient¿s response to therapeutic interventions.
The proposed project aims to: i) improve the existing assays for diagnosis of dysbiosis by development of an LC-MS/MS method to determine urinary concentration of metabolites produced by the intestinal microbiota, which are related with specific subtypes of dysbiosis and, ii) to improve the diagnosis of GI barrier damage, by integration of the standard lactulose/mannitol (L/M) absorption test with a novel assay measuring serum concentration of tight junctions¿ protein, which represent an index of GI barrier breakage. This serum protein test will be developed using a new technology for multiplex detection of analytes in suspension , the PlexBio Precision Imange Code microdiscs with the PlexBio Analyzer.
Furthermore, the developed metabolites/proteins assays will be employed to characterized the dysbiosis/intestinal permeability profile in a group of healthy subjects and in a group of metastatic non-small cell lung cancer patients treated with afatinib, a drug with important GI toxicity. Association between urinary/serum concentration of the analyzed markers will be studied to detect possible relations with the individual risk of GI toxicity during treatment with afatinib and also with the therapy outcome, since metabolic reactions by microbiota and GI barrier damage may affect absorption of orally administrated medications.
The proposed project seeks to introduce two level of novelty in the widely explored area of dysbiosis and GI barrier function, which may have impact on the many clinical areas where these phenomena have been involved, despite their character of causality or consequence of a pathological state.
First, the usually employed assay for dysbiosis and GI function will be implemented by development of sensitive and cost-effective analytical methods to determine further markers, that is:
- dysbiosis diagnosis will be improved by introduction of an LC-MS/MS method to determine (in urine), besides usual markers as skatole and indican, the following products of microbiota metabolism: indole, agmantin, mercaptan, cadaverin, putrescin, tyramine, para-cresyl sulfate, 4-ethylphenylsulfate and short-chain fatty acids. Availability of this methods will allow, in future, to easily perform (LC-MS/MS methods are rapid, very cheap, sensitive and robust) association studies with clinical signs of dysbiosis, patients¿ diet and lifestyle, outcome of intervention procedures, in a variety of medical conditions. The contribution to the advance of knowledge is thought to be paralleled by the implementation of the method in clinical practice.
- Assessment of GI function, i.e. intestinal permeability, will be improved by introduction of a method to determine the serum concentration of tight junction proteins, as an index of GI barrier damage. This determination will be developed using a novel technology for multiplex determination of different analytes in suspension, the PlexBio¿s Precision Imange Code (pcode) microdiscs, thanks to the collaboration with the Euroclone Diagnostia srl (Siziano, PV, Italy), which will furnish the entire equipment (Digiplex Thermocycler, IntelliPlex 1000pCode processor, PlexBio 100 Analyzer) free of charge, aimed to test the potentiality of the instrument to enter the workflow of both research and clinical diagnostics. The validation of a multiplex protein assay in suspension would be of great value to tear down cost per protein analyte (often cost is the limiting step for protein analysis), and will allow to easily introduce novel protein markers in the same assay according to clinical evidences of utility.
Second novelty, the molecular urinary and blood profiles associated with dysbiosis and GI barrier functionality will be studied in relation with both the GI side effects and the outcome of a high impact drug as afatinib. Since the role of metabolic reactions by microbiota and the role of the integrity of the GI barrier in drug efficacy and toxicity are not quite studied, despite their possible high impact on drugs¿ action, methods and results from this study may contribute to establish the research pipeline in the field.