Assessing dehydroepiandrosterone in saliva: a simple radioimmunoassay for use in studies of children, adolescents and adults.

While salivary assays for some hormones are widely used, the availability of assays for salivary DHEA is limited. By adapting a commercially available radioimmunoassay serum kit, we developed a reliable, efficient and sensitive measure of DHEA in saliva that does not require separation or extraction. The minimum detection limit was 4.0 pg/ml. Intra-assay coefficients of variation (CV%) were on average 4.05, and inter-assay CVs averaged 9.70. Method accuracy, determined by spike recovery, and linearity, determined by serial dilution, averaged 99.55 and 92.03%. Levels in matched serum and saliva samples showed strong linear relationships for adult males and females. Specific guidelines are developed for sample collection, storage, and preparation procedures. Reference ranges for salivary DHEA levels are provided for 64 children ages 8-11, 96 adolescents ages 12-17 and 48 adults ages 30-45. Salivary DHEA levels are shown to reflect developmental, gender and diurnal differences.

EGFR-STAT3 signaling promotes formation of malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) develop sporadically or in the context of neurofibromatosis type 1. Epidermal growth factor receptor (EGFR) overexpression has been implicated in MPNST formation, but its precise role and relevant signaling pathways remain unknown. We found that EGFR overexpression promotes mouse neurofibroma transformation to aggressive MPNST (GEM-PNST). Immunohistochemistry demonstrated phosphorylated STAT3 (Tyr705) in both human MPNST and mouse GEM-PNST. A specific JAK2/STAT3 inhibitor FLLL32 delayed MPNST formation in an MPNST xenograft nude mouse model. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR-STAT3 pathway is necessary for MPNST transformation and establishment of MPNST xenografts growth but not for tumor maintenance. Efficacy of the FLLL32 pharmacological inhibitor in delaying MPNST growth suggests that combination therapies targeting JAK/STAT3 might be useful therapeutics.

Assessing salivary cortisol in studies of child development.

In a series of studies, we evaluated the susceptibility of radioimmunoassays (RIA) for saliva cortisol to interference effects caused by oral stimulants used to facilitate saliva collection in studies with children. When added directly to saliva samples, oral stimulants (drink mix crystals) artificially inflated estimated cortisol concentrations. The magnitude of the interference effect was concentration-dependent and more pronounced for some stimulants and RIA procedures than for others. Analysis of samples collected using oral stimulants from child and adult participants confirmed stimulant interference as an extraneous source of variability in measured saliva cortisol. Associations between serum and saliva cortisol and between saliva cortisol and "behavioral" variables were attenuated by stimulant interference. A survey of six large child studies estimated interference effects, indexed by low sample pH, to be present in 14.7% of the 1,148 total saliva samples, or 2%-54% (M = 22%) of samples within each study. Recommendations to minimize the impact of stimulant interference in studies involving salivary cortisol in the context of child health and development are outlined.

Salivary testosterone determination in studies of child health and development.

Measurement of hormones in children's saliva has excited interest because of numerous potential applications in developmental studies. Although assays of children's saliva for some hormones (e.g., cortisol) are widely available and used, the availability and use of assays of children's saliva testosterone is restricted. By adapting a commercially available serum testosterone kit, our laboratory has developed a reliable, efficient, and highly sensitive procedure for measuring testosterone in children's saliva that does not require separation or extraction. The minimum detection limit was 0.8 pg/mL. Intraassay coefficients of variation (CV) were between 3.66 and 6. 78% at concentrations 9.25 to 86.41 pg/mL, and interassay CVs were between 5.70 and 6.61% at concentrations of 7.3 to 118.51 pg/mL. The standard curve was highly reproducible (M slope = -0.70 and Mr = 0. 99). Method accuracy, determined by spike recovery, and linearity, determined by serial dilution, were 99.20 and 92.80%, respectively. Values from matched serum and saliva samples showed strong linear relationships. The assay captured near 99.09% of the range of individual differences in boys' (N = 90) and girls' (N = 85), ages 8-12, am and pm salivary testosterone levels. This assay can be easily applied to the investigation of testosterone-behavior relations in the context of studies on child health and development. It may help many child development researchers improve or expand their research activities.

Assessing estradiol in biobehavioral studies using saliva and blood spots: simple radioimmunoassay protocols, reliability, and comparative validity.

We developed simple, reliable, and highly sensitive assay modifications of commercially available radioimmunoassay kits to measure estradiol in saliva and blood spot specimens. The saliva assay has average intra- and interassay coefficients of variation (CV) of 6.45 and 9.01%, with average analytical and serial dilution recoveries 100.65 and 89.25%. The blood spot assay has average intra- and interassay CVs of 7.57 and 8.22%, with analytical and serial dilution recoveries of 80.50 and 108.50%. The analytical sensitivity ranges of the saliva (0.25-7.50 pg/ml) and blood spot (2. 00-375 pg/ml) assays are sufficient to determine levels in the majority of pre- and postpubertal males and females. Blood spot assay results are correlated with serum estradiol levels for adult males, r (17) = 0.73, and females, r (18) = 0.96. In contrast, the serum-saliva correlation is only modest for adult females, r (14) = 0.60, and not significant for adult males. Substitution of blood spot assay results for serum values underestimates the known serum estradiol-behavior correlation by only 3.45%, whereas substitution of saliva assay results for serum values underestimates the association by 37.55%. The findings have important implications for the use and potential misuse of noninvasive measures of estradiol in studies of health and human development.