Discovery of putative salivary biomarkers for Sjögren's syndrome using high resolution mass spectrometry and bioinformatics.

The purpose of the current study was to determine if saliva contains biomarkers that can be used as diagnostic tools for Sjögren's syndrome (SjS). Twenty seven SjS patients and 27 age-matched healthy controls were recruited for these studies. Unstimulated glandular saliva was collected from the Wharton's duct using a suction device. Two µl of salvia were processed for mass spectrometry analyses on a prOTOF 2000 matrix-assisted laser desorption/ionization orthogonal time of flight (MALDI O-TOF) mass spectrometer. Raw data were analyzed using bioinformatic tools to identify biomarkers. MALDI O-TOF MS analyses of saliva samples were highly reproducible and the mass spectra generated were very rich in peptides and peptide fragments in the 750-7,500 Da range. Data analysis using bioinformatic tools resulted in several classification models being built and several biomarkers identified. One model based on 7 putative biomarkers yielded a sensitivity of 97.5%, specificity of 97.8% and an accuracy of 97.6%. One biomarker was present only in SjS samples and was identified as a proteolytic peptide originating from human basic salivary proline-rich protein 3 precursor. We conclude that salivary biomarkers detected by high-resolution mass spectrometry coupled with powerful bioinformatic tools offer the potential to serve as diagnostic/prognostic tools for SjS.

Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice.

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Genetic LAL mutations lead to Wolman disease (WD) and cholesteryl ester storage disease (CESD). An LAL-null (lal(-/-)) mouse model resembles human WD/CESD with storage of CEs and TGs in multiple organs. Human LAL (hLAL) was expressed in Nicotiana benthamiana using the GENEWARE expression system (G-hLAL). Purified G-hLAL showed mannose receptor-dependent uptake into macrophage cell lines (J774E). Intraperitoneal injection of G-hLAL produced peak activities in plasma at 60 min and in the liver and spleen at 240 min. The t(1/2) values were: approximately 90 min (plasma), approximately 14 h (liver), and approximately 32 h (spleen), with return to baseline by approximately 150 h in liver and approximately 200 h in spleen. Ten injections of G-hLAL (every 3 days) into lal(-/-) mice produced normalization of hepatic color, decreases in hepatic cholesterol and TG contents, and diminished foamy macrophages in liver, spleen, and intestinal villi. All injected lal(-/-) mice developed anti-hLAL protein antibodies, but suffered no adverse events. These studies demonstrate the feasibility of using plant-expressed, recombinant hLAL for the enzyme therapy of human WD/CESD with general implications for other lysosomal storage diseases.

Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes.

Cottontail rabbit papillomavirus (CRPV) and rabbit oral papillomavirus (ROPV) represent distantly related, cutaneous and mucosal tissue tropic papillomaviruses respectively that can infect the same host. These two viruses were used to test the effectiveness of an L2 peptide-based vaccine (aa 94-122) that was delivered on the surface of recombinant tobacco mosaic virus (rTMV) particles. Groups of NZW rabbits received combinations of CRPVL2, ROPVL2 and CRPV+ROPVL2 rTMV vaccines, and were then challenged with infectious CRPV and ROPV. The rabbits developed antibodies that reacted to whole L2 protein and these sera were able to neutralize CRPV pseudovirions at half-maximal titers that were between 50 and 500. Rabbits receiving the CRPV L2 vaccine alone or in combination with ROPV L2 vaccines were completely protected against CRPV infections. Those rabbits vaccinated with the ROPV L2 vaccines showed a weak response in some rabbits against CRPV infection. These studies demonstrate that L2-based vaccines provide strong protection against experimental papillomavirus infection that is most likely based upon the induction of virus-neutralizing antibody. Notably, we observed some limited cross-protection induced by the L2 sequences tested in these vaccines. Finally, the study demonstrated that rTMV were excellent agents for the induction of strong protection in a pre-clinical disease model of papillomavirus infection.