Application of mass spectrometry to elucidate the pathophysiology of Encephalitozoon cuniculi infection in rabbits.

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits.

Cholesterol and glycosphingolipids of human trabecular meshwork and aqueous humor: comparative profiles from control and glaucomatous donors.

PURPOSE: To determine the differential profiles of cholesterol and glycosphingolipid species and their quantitative differences between control and glaucomatous aqueous humor (AQH) and the trabecular meshwork (TM) derived from human donors. METHODS: Control TM and selected primary open angle glaucoma (POAG) TM samples were collected from cadaveric donors. Other TM samples, glaucomatous AQH and control AQH were procured during intraocular surgery. Lipid extraction was performed using modifications of the Bligh and Dyer method. Protein concentration was estimated using the Bradford colorimetric assay. Cholesterol and glycosphingolipids were identified and subjected to ratiometric quantification utilizing precursor ion scan and neutral ion loss scan in positive ion mode using appropriate class specific lipid standards (Cholesterol and Psychosine) on a TSQ Quantum Access Max mass spectrometer. RESULTS: Control and glaucomatous AQH demonstrated 7 and 4 unique cholesterol species, whereas the TM demonstrated 7 and 12 unique species, respectively. The control and POAG AQH showed 6 and 0 whereas TM samples showed 5 and 1 unique glycosphingolipids, respectively. A total of 65 and 62 common cholesterol species and 59 and 58 common glycosphingolipids were found in AQH and TM, respectively. Increased zymosterol and glucopyranosyl cholesterol levels were found in glaucomatous AQH. Significantly decreased levels of galactosylceramide, glucosylceramide in glaucomatous TM were found compared to control TM. CONCLUSION: A high percentage of cholesterol and glycosphingolipid species was found to be common between control and POAG AQH and TM. Several cholesterol and glycosphingolipid species was found to be unique in a subset of POAG or controls. Glaucomatous aqueous humor and TM showed relatively higher levels of zymosterol (an intermediate precursor of cholesterol) and decreased glycoceramide levels, respectively.