Integration of Salivary Bioscience Into Behavioral, Health, and Sports Sciences


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Presentation by Douglas A. Granger at the Building Research Collaborations Retreat, Aug. 23, 2012

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Integration of Salivary Bioscience Into Behavioral, Health, and Sports Sciences

  1. 1. Integration of Salivary Bioscience intoBehavioral, Health, and Sports Sciences: How Biomarkers in Saliva can Benefit Your Research Program Douglas A. Granger, Ph.D. Center for Interdisciplinary Salivary Bioscience Research Johns Hopkins University
  2. 2. Disclosure StatementIn the interest of full disclosure, Douglas A. Granger is thefounder (or co-founder), serves as the Chief Scientific andStrategy Advisor, and holds equity in Salimetrics LLC andSalivabio LLC.These relationships are managed by the policies of theConflict of Interest Committee at the Johns HopkinsUniversity School of Medicine
  3. 3. Overarching AssumptionsEffects of context on development moderatedthrough individual differences in stress responsivebiological systemsBiology and behavior have reciprocal effects andexpression of bio-behavioral relationships isdependent on context Probability that individual differences in biological reactivity and regulation linked to outcomes of interest highest when studied in meaningful social contexts Biological systems are networked and multi-system measurement of stress response is critical
  4. 4. Perturbing the organism: The biology of stressful experienceEnvironmental Demands(physical, social, cultural)Behavioral Surface(emotion regulation, coping,flight-fight, tend-befriend)Fast Acting—Physiological Processes(neural, HPA, ANS activity)Slow Acting—Physiological Processes(genetic activity) Individual Development Biological Susceptibility to Context, Adaptive Calibration Weiner (1992) Gottlieb (1992) Boyce and Ellis (2005)
  5. 5. Intra-individual patterns of stress-reactivity Normative-adaptive pattern assumes habituation of 6 reactivity and recovery over time 5 4 3 2 1 0 1 2 3 4 5 Exposure historyTimeMcEwen
  6. 6. Advantages of Oral Fluid as Research Specimen ____________________________________________________________________________________ “Minimally Invasive” Considered “acceptable and non-invasive” by research participants Collection is quick, non-painful, uncomplicated _____________________________________________________ “Safety” Reduces transmission of infectious disease by eliminating the potential for accidental needle sticks CDC does not consider saliva a class II Biohazard unless visibly contaminated with blood ______________________________________________________ “Self-collection” Allows for community- and home-based collection Enables specimen collection in special populations ______________________________________________________ “Economics” Eliminates the need for a health care intermediary (e.g., phlebotomist, nurse). Resources for collection and processing samples are low cost and available _____________________________________________________ “Accuracy” Salivary levels of many analytes represent the “free unbound fraction” or biological active fraction in the general circulation _____________________________________________________ “Ecological Validity” Enables biological reactivity and regulation to be monitored in everyday social world _____________________________________________________ “ Multiple Participants” Enables samples to be collected from groups of individual participants simultaneously in real timeSlavkin /Mandel/Malamud
  7. 7. Oral fluid subtypes that compose “Whole Saliva”  Sublingual saliva (4%)  Parotid saliva (23%)  Submandibular saliva (65%)  Minor saliva glands (7%)  Cervicular fluid (1%)Mandel
  8. 8. Movement of Biomarkers into Saliva Acinar cells Passive diffusion Ultrafiltration transport proteins Active transport Synthesiscapillariessurroundingglands
  9. 9. Cortisol is secreted into the bloodstream from the adrenal cortex. Adrenal Cortex About 95% of it binds to carrier proteins (binding globulin). Only the remaining 5% is available for use by target tissues. Numerous capillaries surround the saliva glands. Hormones and other compounds pass through the capillary walls and bathe the salivary glands. Secretory cells of the saliva glandsBlood Saliva Unbound cortisol is lipid soluble and can move through the lipo-protein cell membranes of the secretory cells by passive diffusion. It is then released into the saliva.
  10. 10. Many analytes are released locally into oral fluids and are not from bloodKnowing whether an analyte measured in oral fluid is or is not from blood is key to interpretation of the meaning of individual differences
  11. 11. Analytes in Oral Fluid of Interest to Health Sciences cytokines Cortisol Dehydroepiandrosterone (s) Testosterone, androstenedione Estradiol, estrone, estriol Antibodies sIgA Progesterone and 17-alpha-hydroxy-Progesterone Cotinine Drugs of Abuse and Environmental Chemicals C-Reactive Protein, Neopterin, Beta-2-microglobulin Melatonin, Oxytocin, NPY, VIP Cytokines (e.g., IL-6, TNFa, IL1b), soluble cytokine receptors (sTNF-I, -II) Disease specific antibodies (e,g, HIV, HSV, EBV, CMV) or antigens Alpha-amylase, Chromogranin A Metalloproteinases (MMP-8) DNA Heat Shock Proteins, aldosterone Adiponectin, Leptin, Ghrelin Electrolytes DNA and Methylation, RNA Microbiome GFAP, Neurogranin
  12. 12. Collection Device Aid Mouth Piece and Flute Treatment Chamber Vented connector to storage vial
  13. 13. Salivette Module with Oral SwabEasy to useExcellent Recovery1.5 mL capacity Restricted analysis potential Small size-choking hazard Large head space Location of placement
  14. 14. BD Hydrocellulose MicrospongeHandleHighly absorbentRecovery of low volumesSleeping participant300 uL capacity High surface area Evaporation hazard Use two Restricted analysisHarmon et al (2007). Hormones and Behavior.
  15. 15. Handling, Transport, Storage Biosafety Bacteria growth and specimen stability Freeze thaw Field storage Shipping rules and regulations Long term storage and archiving
  16. 16. Enzyme Immunoassay Competitive Binding
  17. 17. Multiplexing Technology
  18. 18. Dampened Salivary Alpha-amylase and Cortisol Reactivity to Psychosocial Stress : Maltreated versus Comparison Adolescents 4.50 160 4.00 140 3.50 120 Salivary AA (U/mL) 3.00 Cortisol (nmol/L) 100 2.50 80 2.00 60 1.50 Cortisol (Comparison) Cortisol (Maltreated) 40 1.00 Salivary AA (Comparison) Salivary AA (Maltreated) 20 0.50 0.00 0 5) 5) 5) 0) ) 35 5) +1 +2 +4 -1 -4 + = = = = = = (T (T (T (T (T (T ss 10 30 ss n 20 tio re re + + + St St xa ss ss ss e la st re re Pr re Re Po St St St e st st st Pr Po Po PoGordis et al (2008) Psychoneuroendocrinology
  19. 19. Cortisol, Alpha-amylase, and subjective emotional reactivity in women with Borderline Personality Disorder (BPD) (Scott, Granger, Levy, 2011). The BPD group showed attenuated stress-related cortisolreactivity as compared to both NTM and TM groups. The BPD group had higher average baseline and overallBoth the BPD and TM groups demonstrated attenuated stress- average NA than both NTM and TM groups.related sAA reactivity as compared to the NTM group.
  20. 20. Developmental differences in sAA Response to Inoculation Stress 2 Months 100 6 Months  2-month old infants did not 12 Months Infant Alpha Amylase U/ml 80 24 Months show an sAA response 60  6 and 12-month old infants 40 displayed a significant sAA increase. 20 0  24-month old infants Baseline 5 Min 10 Min 20 Min displayed an anticipatory Time rise and decrease in sAA InoculationDavis & Granger (2009). Psychoneuroendocrinology, 34, 795-804
  21. 21. Internalizing problem behavior and sAA reactivity to social exclusion task Lo Internalizing Problems Hi Internalizing Problems 220 180 Saliva AA 140 100 60 1 2 3 4 5 6 Interact 1 Interact 3 Interact 2Allwood et al., (2011). Biological Psychology, 88, 57-64
  22. 22. Maternal Engagement in Early Infancy Predicts Children’s Cortisol Reactivity at 15 months Blair et al, (2008) Developmental Psychology, 44, 1095-1109
  23. 23. Effects of early adversity on stress reactivity in adulthoodBirth Group Neonatal CriteriaFull Term Full Term; medically & neurologically healthyHealthy Preterm Premature no medical/neurological complicationsMedical Preterm Premature neonatal medical illness (BPD, RDS, NEC, sepsis)Neurological Preterm Premature neonatal neurological illness (Grade III & IV IVH, meningitis, shunted hydrocephalus)Small for Gestational Age PrematurePreterm birth weight for gestational age < 10th percentileFull Term (>37 weeks gestation) Premature (<37 weeks gestation) Sullivan, Mills, Winchester, Granger (in prep) 23
  24. 24. HPA Stress Reactivity In Adulthood by Birth Group 0.5 HPT group: : most dysregulated NPT group: highest post-stressor 0.45 pattern for stress reactivity cortisol levels, with the sharpest 0.4 decline. Recovery doesMean Raw Units of Cortisol not fall below baseline 0.35 0.3 FT HPT 0.25 (ug/dL) MPT 0.2 NPT 0.15 SGA SGA group: blunted pattern marked by lower 0.1 than normal baseline, lower peak cortisol 0.05 levels 15-minutes post-stressor, and a slow recovery to social stress 0 baseline 1 baseline 2 swab 3 swab 4 swab 5 swab 6 swab 7 swab 8 1:00pm 1:30pm 1:47pm 2:00pm 2:15pm 2:30pm 2:45pm 3:00pm After TSST Cortisol Ends Recovery Intro Peaks TSST 7.20.2012 Starts 24
  25. 25. Army Nurse Combat Casualty Stress Task McGraw et al. (2012) Psychoneuroendocrinology
  26. 26. Salivary Cortisol Response to Rowing Ergometer Competition Men and women’s endocrine responses were more different than alike and varied by level of experienceIndividual differences associated with socialAfiliation rather than dominance orcompetitiveness Kivlighan, Granger, Booth (2005). Psychoneuroendocrinology
  27. 27. Salivary sAA Response to Rowing Ergometer CompetitionsAA higher for varsity than novice,and associated with performance.sAa reactivity associated withperceived dominance sAA reactivity specific to competition, and levels Higher in men than women Kivlighan & Granger (2006) Psychoneuroendocrinology
  28. 28. Mucosal IgA and URTI in American College Football Players: A Year Longitudinal Study 100 160 Footballers Controls 140 80 120Percent with URTI Saliva IgA (mg/L) 60 100 80 40 60 Footballers 40 Controls 20 20 0 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Stage of season Stage of season Autumn Winter Spring Fahlman & Engels (2005) Med Sci Sports Exerc 37(3): 374-380
  29. 29. Immunological differences in English Premier League football players with infections 28 players in total were sampled on up to 4 occasions (total of n=94); 5 players had viral infections on 11 occasions 2.5 300 P<0.01 2 P<0.01 TH/TC (CD4/CD8) Ratio 250Saliva IgA (mg/L) 1.5 200 150 1 100 0.5 50 0 0 Healthy With Infection Healthy With Infection Gleeson
  30. 30. A longitudinal study of changes in s-IgAand respiratory illness in athletes• 38 members of America’s Cup yacht crew• Studied over 50 weeks of training and sailing• Morning saliva samples collected weekly• Clinically confirmed illnessNeville, Gleeson & Folland (2008) Med Sci Sports Exerc 40(7):1228-1236
  31. 31. Relative s-IgA before and after an infection episode Gleeson
  32. 32. Conclusions• On a group basis, relative s-IgA determined a substantial proportion of the variability in weekly infection incidence• Significant reduction in s-IgA in the 3 weeks prior to infection• Relative s-IgA value <40% of healthy baseline value indicated a 50% chance of contracting an infection within 3 weeksHence, possible predictive value with regular monitoring Gleeson
  33. 33. Monitoring hormones With stress: C and T C/T ratio 30% = Overreached/Stressed Salivary C/T (1000*nM/pM) ratio > 40 = Stressed or Not Recovered
  34. 34. Monitoring recovery in RFU players 10 England International RFU Players 29 1 years, 102.4 3.9 kg, 1.86 0.02 m 4 saliva samples collected each wk for 3 weeks Morning collection, before breakfast IgA, Cortisol, Testosterone measured by ELISA Transferrin (blood contamination) by ELISA Training load monitored 3 international matches Players’ perceptions of fatigue and stress monitored using the REST-Q questionnaire Gleeson
  35. 35. Training Load 0 1 2 3 4 5 6 7 8 9 10 30/10/2006 31/10/2006 01/11/2006 02/11/2006 03/11/2006 04/11/2006Gleeson 05/11/2006 06/11/2006 L 20-41 07/11/2006 08/11/2006 09/11/2006 10/11/2006 11/11/2006 12/11/2006 L 18-25 13/11/2006 14/11/2006 15/11/2006 16/11/2006 17/11/2006 18/11/2006 W 23-21
  36. 36. Cortisol / Testosterone Ratio 0 10 20 30 40 50 60 70 30/10/2006 #1 31/10/2006 01/11/2006 * 02/11/2006 03/11/2006 04/11/2006 05/11/2006 L 20-41 06/11/2006 07/11/2006 *P<0.05; **P<0.01 vs #1 ** 08/11/2006 09/11/2006 ** 10/11/2006 ** 11/11/2006 L 18-25Effect of time (P<0.001) 12/11/2006 13/11/2006 * 14/11/2006 15/11/2006 16/11/2006 17/11/2006 18/11/2006 W 23-21
  37. 37. Conclusions• On a group basis, saliva C/T ratio appears to be indicative of stress or inadequate recovery• May help in recovery prescription for individual players• S-C/T >40 likely associated with underperformanceHence, possible value with monitoring during intensive phases of season Gleeson
  38. 38. Momentary Cortisol - Behavior Associations in Everyday Contexts 0.6 Diurnal variation determined by fixed Momentary Determination sampling at specific times 0.5 * 0.4 Residual above curveCortisol ug/dL 0.3 0.2 0.1 Residual below curve 0.0 Wake 30 Mins Noon * Evening -0.1 Time Momentary Determination
  39. 39. Dyadic Similarity in Cortisol Levels Father Middle Child Mother Mother Father .34** (326) Middle Child .19** .21** (320) (326) Adolescent .34** .24** .22** (320) (322) (318) ** P<.01400 Mothers, Fathers, Older and younger siblingSaliva sampled at home two morningsRelationship Quality ModeratesMartial satisfaction and conflict, spouse who are congruent havehigher covariation in cortisol levelsBooth, Johnson, Goslin, & Granger ( in prep)
  40. 40. Commonality among members of social groups Family LevelLatent Level Construct * Father Sibling BManifest Level Mother Sibling AApproach would theoretically enable the exploration of factors that contribute toeach individual members contribution to the family level construct, and contribute todifferences between groups.
  41. 41. Social Networks: Families, Teams, ClassesSelf-reported Friendship Nominations in 6th Grade Thick blue lines are reciprocated Friendships Thin grey lines are asymmetric FriendshipsData provided by Scott Gest, PhD, Penn State University
  42. 42. Cortisol, Testosterone, and the Social Network: Friendship Nominations Cortisol Testosterone
  43. 43. Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples.Are the typical volumes of saliva collected in studies of child development sufficient to enable isolation of DNA for genetics analyses?Do collection device materials affect the isolation of sufficient quantity and quality of DNA from saliva?What are the effects of room temperature (RT) storage of saliva samples on the quality and quantity of DNA recovered?Do archived saliva samples that have been exposed to multiple freeze-thaw cycles yield sufficient quantity and quality of DNA for genetic analyses?When oral fluids are collected from specific areas in the mouth associated with different salivary glands, is there a difference in DNA quantity and quality? Nemoda, et al. (2012). BMC Medical Research Methodology.
  44. 44. OXTR and the Social Network: Friendship Nominations AA – Black; AG – Red; GG - Blue
  45. 45. Epigenetics Global Methylation -- Levels correlated in DNA extracted from cells from oral fluid compared to DNA extracted from cells from whole blood
  46. 46. Cytokines Design: 113 Healthy Adolescent Girls, multiplex EC Immunoassay, 3 waves of annual samplings GM-CSF, IFNγ, IL-1ß, IL-2, IL-6, IL-8, IL-10, IL12-p70, and TNFa. Cytokine levels: all cytokines, except IFNγ and IL-10, were detectable in the majority of saliva samples; non-normal distributions; levels of all cytokines lower in saliva than serum except IL-8 and IL-1ß; saliva levels of IL-8 and IL-1ß were >20x higher than other salivary cytokines; Cytokine Inter-correlations : in saliva were lower than in serum; individual differences in cytokine levels were more stable across years when measured in serum than in saliva; Stability : there were strong associations between salivary adiponectin and cotinine with salivary but not serum cytokines; and Serum-saliva correlation: only IL-1ß Correlates: Salivary cytokine levels were not associated with age, pubertal development, or gynecological age, Conclusion: Variation in salivary cytokine levels largely reflects compartmentalized local activity of the oral mucosal immune system raising questions about the role they play, if any, in intracellular and hormonal signaling within the neuro-endocrine-immune network. Granger et al (in prep)
  47. 47. Prospective Markers of TBI in Saliva Name Type Primary Source Primary Function/Use Size filament and marker of astrocytes, GFAP cytoskeleton astrocytes astrocyte differentiation, 45 kDa protein astrogliosis, tumor marker participates in intracellular cytoplasm of S100B calcium-binding activities, marker of astroglia and 20 kDa protein pathological conditions and Schwann cells injury cytoplasm of released under pathological neurons, NSE glycolytic enzyme conditions, marker of cell 78 kDa neuroendocrine damage, tumor marker cells participates in synaptic neuroprotein, signaling, marker of dendrite Neurogranin protein kinase C neurons 7.5 kDa spine density and synaptic substrate plasticity marker related to neuron BDNF neurotrophin widespread in CNS 14 kDa growth and survival development and presynaptic brain Beta-Synuclein soluble protein maintenance of synaptic 19 kDa tissues, astrocytes function, tumor marker synapse development, transmembrane ICAM-5 neurons immune and inflammatory 130 kDa glycoprotein responses protects neurons and other metal-binding MT-III astrocytes tissues against toxic factors, 6 kDa protein oxidative stress
  48. 48. Future Directions Salivary bioscience is poised to enable and facilitate advances in multiple fields Emerging applications have implications for screening and diagnostics, and decisions related to personal health, well being, and performance Prevention and intervention science Community of interdisciplinary scholars linked together by an international network of centers of excellence
  49. 49. Questions and Discussion Johns Hopkins University Center for Interdisciplinary Salivary Bioscience Research