Saliva as a diagnostic fluid

1. Saliva as a diagnostic fluid DENT 5302 Topics in Dental Biochemistry Dr. Joel Rudney

2. Why use saliva? • Collection is relatively non-invasive - no needles! • Range of potential analytes as broad as plasma • Bacteria in saliva • Salivary ions • Secreted salivary proteins • Inflammatory mediators • Proteins, metabolites, from other parts of the body • Nucleic acids (from the mouth and other regions) • This is considered a high-priority research area • The vision - chairside diagnostic testing

3. The search for biomarkers • A big buzzword in diagnostics today • Any sort of analyte can be a biomarker • The keys to the definition • Changes in a biomarker must be associated with the active presence of a disease, or future risk of a disease • There doesn’t have to be a direct etiological relationship • A marker for risk or diagnosis • Multiple analytes can improve sensitivity/specificity

4. Earlier efforts - caries risk • Bacteria in saliva as biomarkers - chairside tests • Dentocult SM® "Strip mutans", S. mutans in saliva • Dentocult LB®, Lactobacilli in saliva • Dentobuff® test strips, salivary buffering capacity (HCO3-) • General problem - high sensitivity, but low specificity • Combination testing (all three plus flow rate) • Not much improvement • These products not widely used in the U.S.

5. Other oral diseases • Detect candidal infection - Oricult N® • Not widely used in the U.S. • Periodontitis • The major focus is on biomarkers in gingival fluid • Periodontists want to predict which sites will lose attachment • Saliva can only provide indirect indications

6. Saliva drug testing • Main focus is drugs of abuse • Much less invasive than observed urine testing • Harder to fake, no oral equivalent of the “whizzinator” • Products for “roadside” forensic testing • Rapiscan ®, Oral-Screen™, 4-5 drugs simultaneously • Cannabinoids, opiates, meth, cocaine, diazepines • Some published validation of these methods • Largely unregulated online offers for home testing • Matched by online offers of products to beat the tests • No real validation of either

7. Saliva and steroids • Passively carried into saliva with water from plasma • Accurately correlated with plasma levels • Monitoring of female reproductive hormones • Convenient for monitoring therapeutic uses • Post-menopausal HRT • Menstrual cycle timing • Saliva and androgens • Can be used to monitor testosterone levels • HRT, but also by bodybuilders • Steroids of athletic abuse (with the right test system) • Also lots of online activity

8. Saliva and HIV testing • Extremely low levels of virus relative to other body fluids • Assumed to be due to anti-viral salivary proteins • SLIPI, MUC7 • Antibodies to HIV (protective or not?) are present • The basis for HIV testing in saliva • Saliva-based home HIV test on track for approval • Controversial issues • Pro - more people will get tested, seek treatment • Con - no counseling, outside the public health system

9. Sjogren’s Syndrome proteome • Early detection of salivary gland changes • Previous studies of individual proteins • Differences, but sensitivity/specificity inadequate • Can this be improved by comparing proteomes? • Recent data from NIDCR • Compared to controls, patients show: • Incr. inflammatory proteins: lysozyme, lactoferrin, cystatin C, 2-microglobulin, Ig -light chain, polymeric Ig receptor (component of S-IgA) • Decr. acinar proteins:presumed PRPs, amylase, carbonic anhydrase • Key question: How early do these changes begin?

10. Cancer antigens in saliva • Breast cancer biomarkers • Salivary c-erbB-2 protein - an oncogene product • Elevated in breast cancer patients • Sensitivity = 87%, specificity = 65% • Proteomic studies in progress • Not useful for prostate-specific antigen (PSA)

11. Saliva and oral cancer • The earlier the diagnosis, the better the prognosis • Dentists are the first line of defense • Oral cancer is hard to distinguish from transitory lesions • When should you biopsy? • Can pre-cancerous conditions be detected? • Can salivary biomarkers provide an earlier warning? • An area of active research at U of M

12. Many potential biomarkers • Elevated levels of certain bacteria in saliva • Three very common commensal species • Capnocytophaga gingivalis, Prevotellamelaninogenica, and Streptococcus mitis • Probably effect rather than cause • Sensitivity = 80%, specificity = 82% • Proteomes, genomes, and transcriptomes • Dr. David Wong at UCLA School of Dentistry • Collaborators at U of M • Drs. Nelson Rhodus and Patrick Gaffney • Dr. Kathy Moser (Sjogren’s)

13. Findings from the Wong lab • Oral cancer biomarkers in the salivary proteome • Elevated levels of the proinflammatory cytokine IL8 • A general marker of inflammation • Other sources of oral inflammation - confounders? • Additional protein biomarkers may be needed • Oral cancer biomarkers in the salivary transcriptome • Wong lab discovered that saliva contains “intact” mRNA • Marker of changes in gene expression due to cancer? • Seven genes upregulated in patients relative to controls • Includes two proinflammatory cytokines, IL8 and IL1

14. Chairside testing • Wong lab is collaborating with engineers • Objective: “lab on a chip” • Miniaturized multi-analyte detection and quantification • Intended for use in a dental office • Results at chairside • Application: Identify patients to watch or biopsy • Detect oral cancer while it is still treatable • The same concept can be applied to other diseases • Oral and systemic diseases both • People see dentists more often than physicians • The hunt for biomarkers is ongoing

16. Format of the saliva midterm • Short-answer questions • How Dr. Rudney defines a short answer: • When I say short, I mean short • Some of them can be answered with a single word. • Respond with words, phrases, or diagrams, as seems appropriate • Don’t mess around with complete sentences or essays. • Use only the space provided for each question. • In the case of questions worth more than one point: • The number of points given will be based on the number of correct answers you provide for that question • When I ask for examples, it means that the number of potentially correct answers is greater than the number of examples that I’m asking for. • There are no trick questions (at least, not intentionally)

17. A sample one-point question • Q. Identify the component of the water secretion system that requires energy to function. (1 pt.)

18. A sample one-point question • Q. Identify the component of the water secretion system that requires energy to function. (1 pt.) • A. Na+-K+-ATPase • You get one point

19. A sample multi-point question • Q. Give three examples of proteins found in pellicle (3 pts.)

20. A sample multi-point question • Q. Give three examples of proteins found in pellicle (3 pts.) • A. Statherin, acidic proline-rich proteins (aPRP), histatins • You get three points

21. A sample multi-point question • Q. Give three examples of proteins found in pellicle (3 pts.) • Acceptable answers: • Statherin, acidic proline-rich proteins, amylase, histatins, cystatins, MUC7 mucin, lysozyme, albumin, carbonic anhydrase • Use any three, and you get three points

22. A sample multi-point question • Q. Give three examples of proteins found in pellicle (3 pts.) • Acceptable answers: • Statherin, acidic proline-rich proteins, amylase, histatins, cystatins, MUC7 mucin, lysozyme, albumin, carbonic anhydrase • Your answer: • Statherin, aPRP, basic PRP • You get two points