Hydrocortisone

After arriving at one of the three participating sites, respondents were escorted by project staff to the clinic where they were checked in, and were then escorted to the room where they would stay overnight. In most cases, respondents arrived mid-afternoon of Day 1 of their visit and ended their stay by noon of Day 2. On Day 1, with staff assistance, they completed the medical history, the bone densitometry scan, and physical exam, each of which required 30–45 minutes. They were also given the self-administered questionnaire (SAQ) to complete that evening (see www.midus.wisc.edu for copies of assessment instruments, which are included under descriptions of the MIDUS II projects) Clinic nursing staff began collecting the 12 hour urine specimen (collection period 7 p.m. to 7 a.m.). On Day 2 nursing staff collected the fasting blood specimen and completed the 12 hour urine specimen collection.
After breakfast, project staff carried out an experimental protocol assessing physiological response to, and recovery from, cognitive and orthostatic challenges similar to stressors people experience in their daily lives. The protocol consisted of a series of two randomized 6 minute cognitive challenges, one involving a math task and the other a Stroop-like test (decision-making about stimuli in which letters and colors are in conflict), followed by a 6 minute orthostatic (standing) challenge. Each challenge was followed by a 6 minute recovery period. Physiological reactivity throughout the experimental protocol was monitored via measures of blood pressure, heart rate variability and respiration, and salivary cortisol. Completed SAQs were then collected, and respondents were debriefed. At the UW-Madison data collection site, information was given about completing objective sleep assessments, to be returned by mail, after returning home. At the end of their visits, respondents were given a report about their blood pressure, body mass index (BMI), and waist-hip ratio. They were sent letters reporting cholesterol, HAlc, and bone density 1–2 months after the clinic visit.
To ensure consistency across sites and optimize the pace and quality of data collection, project staff and clinic nursing staff at all three sites followed standardized procedures that were detailed in a general Manual of Procedures, as well as more specific Guidelines for Collecting and Processing Biomarkers, and a Psychophysiology Manual. An administrative database was used to facilitate management and tracking of cross-project participation as well as tracking of participation at the three Project 4 sites. This information allowed review of participation information and quality control assessments, including identifying areas where additional staff training was required. Monthly conference calls with staff and investigators from all sites provided a forum to discuss issues or problems. Prior to these calls, each site generated a “Progress Report”, using report queries built into the administrative database; the reports were circulated for review by all on the conference call.

Primary DMBA induced mouse OSCC were generated as described (26 (link)). Single cell suspensions of individual primary oral cavity tumors were made with Collagenase IA (Sigma-Aldrich) and cultured in IMDM/F12 (2:1) with 5% FCS, penicillin/streptomycin, 1% amphotericin, 5 ng/mL EGF (Millipore), 400 ng/mL hydrocortisone, and 5 μg/mL insulin. Sequential differential trypsinization was then used to clear fibroblast contamination. MOC1, 7, 10, 22 and 23 were derived from primary tumors in C57BL/6 WT mice and MOC2 was derived from a chemokine receptor CXCR3deficient mouse on a pure C57BL/6 background (27 (link)) (of note, no major differences in the incidence of tumor formation were noted between the different genotypes). CXCR3 is not detectable on oral keratinocytes and does not contribute to MOC2 growth (Figure S3). Immunofluorescence staining for cytokeratin was performed to confirm an epithelial phenotype (Figure 1C and Figure S1C). PCI-13 was obtained from Dr. Theresa Whiteside, UPCI:SCC029B and UPCI:SCC068 were obtained from Dr. Suzanne Gollin, and all were used with minimal passaging. The UM-SCC-1 cell line (from Dr. Tom Carey) was genotyped in May, 2011 and concordance with published data was established (27 (link)).