Sharon DiRienzo

Inflammatory Bowel Disease--comprised of Crohn's Disease and Ulcerative Colitis (UC)--is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.

The antibacterial properties of lysozyme were investigated with oral microorganisms representing the seven serotypes (a through g) of Streptococcus mutans, Veillonella alcalescens, and the virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14. Growth of bacteria in defined medium was monitored spectrophotometrically after the addition of various amounts (25 mug to 5 mg/ml) of enzyme. No growth inhibition of V. alcalescens was observed. Inhibition of A. viscosus T14(V) and A. viscosus T14(AV) occurred with 160 mug of lysozyme per ml. Of the S. mutans cultures tested, the serotype a and b strains were inhibited with as little as 25 mug of enzyme per ml, whereas e and f strains were most resistant to the bacteriostatic activity of lysozyme. The presence of dl-threonine or sucrose in growth medium did not significantly affect the results. A lysoplate assay was developed to rapidly survey the bacterial cultures for their susceptibility to the lytic ability of the enzyme. Lysis, as a measure of a zone of clearing in agarose plates, occurred for all microorganisms in the presence of lysozyme after the subsequent addition of NaCl or detergent. The bactericidal activity of lysozyme was determined on S. mutans BHT and S. mutans LM-7 by the pour plate technique. Preincubation of S. mutans LM-7 with as much as 1 mg of enzyme for 90 min did not affect viability or growth, whereas preincubation of S. mutans BHT with 1 mg of lysozyme resulted in no recoverable colony-forming units. An antigen containing extract of S. mutans LM-7 blocked the growth inhibitory property of lysozyme. Human lysozyme was a more effective antibacterial factor than hen egg white lysozyme. Total growth inhibition of S. mutans BHT was effected with 40 mug of human enzyme, and as little as 10 mug of human enzyme inhibited growth for greater than 20 h. The data presented indicate that different mechanisms may be responsible for the bacteriostatic, lytic, and bactericidal properties of the enzyme and that lysozyme is a selective but effective antibacterial factor for oral microorganisms.