Isolation and identification of hydroxyl-platelet-activating factor from natural sources.

Platelet-activating factor (PAF), a potent inflammatory mediator that has previously been detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid (GCF) and in saliva, is implicated in periodontal disease. The biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In a preliminary study this bioactive molecule was detected for the first time in human blood derived from volunteers with chronic periodontitis as well as from periodontally healthy volunteers. Compounds isolated from natural sources as well as synthetic ones have been reported as biologically active lipids with physiological importance based on the fact that they induce platelet aggregation with EC50 values ranging from 100 to 0.01 microM through interaction with G-protein-coupled receptors like the PAF receptor, leading to altered signal transduction. In this study, the existence of hydroxyl-PAF analogue in human blood was further studied as well as its distribution in plasma and in blood components. The existence of hydroxyl-PAF analogue was also investigated in samples from rabbit blood hen's egg yolk. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray MS analysis. Quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of plasma hydroxyl-PAF analogue levels to plasma PAF levels in volunteers with periodontitis. Moreover, hydroxyl-PAF analogue was also detected in rabbit blood and hen's egg yolk samples. These data support that this bioactive lipid may play a role in oral inflammation and suggest PAF as a member of a lipid molecule family with different structures and from different sources which share the same or similar biological activities, apparently with different physiological roles in human and animals.

Immunological and molecular detection of human immunodeficiency virus in saliva, and comparison with blood testing.

In order to test the detection feasibility of human immunodeficiency virus (HIV) in saliva, a three-method blind screening analysis was conducted. Sixty-eight individuals were studied, comprising 34 HIV carriers and 34 noncarriers (controls) of matched gender and age. An oral examination preceded saliva and blood sampling of studied individuals. All samples were tested blind for HIV by using two immunological methods [Oraquick-compatible enzyme-linked immunosorbent assay (ELISA) and a fluorescent immunoenzymatic method (ELFA)], confirmed by western blotting, and a simple molecular method (polymerase chain reaction amplification of a relatively constant viral DNA region), confirmed by DNA hydridization. Compared with the controls, about twice as many HIV carriers had oral health problems, including periodontal disease. ELFA resulted in 33/34 positives and 34/34 negatives in saliva, while it detected 34/34 positives and 34/34 negatives in blood. ELISA performed even better, with correct assignment of all positives and negatives in both saliva and blood. The PCR method, at three annealing temperatures, surprisingly detected all positive samples, while it gave no false-positive result. In conclusion, the detection of anti-HIV in saliva may achieve accuracy of 97.1-100%, comparable with that in blood. Furthermore, this study suggests that a highly accurate molecular method of HIV detection may be feasible, although the studied carriers had rather homogeneous characteristics.

Hydroxyl-platelet-activating factor exists in blood of healthy volunteers and periodontal patients.

Periodontal diseases are localized chronic inflammatory conditions of the gingival and underlying bone and connective tissue. Platelet-activating factor (PAF), a potent inflammatory phospholipid mediator that has been previously detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid and in saliva, is implicated in periodontal disease. Our results from previous studies showed that the biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In this study, hydroxyl-PAF analogue was detected for the first time in human blood derived from patients with chronic periodontitis as well as from periodontally healthy volunteers. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray analysis. In addition, the quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of hydroxyl-PAF analogue levels to PAF levels in periodontal patients, suggesting that this bioactive lipid may play a role in oral inflammation.