Participants and Study Overview
Participants provided written informed consent in accordance with the Declaration of Helsinki. Participants were drawn from a randomized controlled trial examining effects of an 8-week Mindfulness Based Stress Reduction (MBSR) intervention (compared to a waitlist control condition) in adults aged 18 to 60 reporting chronic psychological stress. Participants were recruited from a university campus and surrounding community in a rural region of the southern United States. A full description of the clinical trial and affective outcomes have been reported elsewhere (Carlton, et al., 2021). The current manuscript reports only the methods and results pertinent to (1) the standardization of the PI-MED erythema response across skin pigmentations and PI-MED erythema reliability and (2) associations between PI-MED erythema and covariates. Exclusion criteria for the clinical trial were: extensive previous meditation experience, current daily practice with mind-body techniques such as yoga, any medical conditions or current use of medications linked to disruptions in inflammatory response (e.g., diabetes, use of steroids or corticosteroids), or a self-reported problem with drugs or alcohol. Additionally, individuals taking psychotropic medications were excluded if they reported any changes to their medication in the previous three months, and any participants already taking psychotropic medication were discontinued in the study if they altered their medication during the study. While recruitment materials targeted stressed adults, there were no inclusion/exclusion criteria related to stress levels.
To assess erythema response, PI-MED testing was conducted at baseline (prior to randomization into either the mindfulness intervention or waitlist condition) and at study end-point (approximately 8—10 weeks after baseline assessments and after completion of the intervention/waitlist period). Potential covariates of inflammation were collected at baseline including non-affective covariates (e.g., age, body mass index) and self-report measures of affect. At baseline, sixty-one participants completed PI-MED, however PI-MED data were not collected from 18 participants at end-point (end-point n = 43) for the following reasons: 7 participants withdrew from the study; 5 participants had scheduling conflicts that interfered with end-point data collection; and, out of an abundance of caution, the PI-MED procedure was not conducted on 6 participants due to residual discoloration from PI-MED implemented at baseline (approximately 8-10 weeks prior), or because the participant opted out. See Table 1 for participant demographics.
Precision Implementation of Minimal Erythema Dose (PI-MED). Cutaneous erythema was induced and measured using PI-MED testing, a procedure recently adapted by Richey and colleagues (2019) to precisely measure individual differences in erythema response to ultraviolet (UV) light exposure. The PI-MED procedure is an adaptation of Minimal Erythema Dose (MED) testing (Heckman, et al. 2013) intended to optimize the procedure for reproducibility and investigating individual differences in cutaneous inflammation. See Supplemental Method for a extended rationale for the PI-MED procedure compared to typical MED testing.
Details of PI-MED Standardization of UV-Irradiation. In accordance with procedures described in detail by Richey and colleagues (2019), six small portions of skin on the non-dominant inner forearm were exposed, in sequence, to different timed amounts of UV light, utilizing a novel dosage schedule (Richey, et al., 2019). The novel PI-MED dosage schedule provided by Richey and colleagues (2019) is calibrated to individual differences in skin pigmentation (as described below) to reliably produce erythema across the Fitzpatrick Skin Types (Fitzpatrick, 1988). Specifically, the dosage schedule provides a method for standardizing UV dosage under a constant assumption of UVB energy at 270 μW/cm2, which is monitored via handheld radiometer. Based on published median dosages (Gambichler, et al., 2006) required to produce a minimal erythema response for skin types II, III, and IV, the PI-MED dosage schedule was extrapolated to capture the minimal erythema response at the mid-point of a given dosage schedule for each skin type by assuming a linear relationship between skin type and dosage needed to produce a minimal erythema response (see Richey, et al., 2019 for further details). This calculated dosage schedule, in combination with measurement of erythema via instrumentation (handheld spectrophotometer, as below) was intended to provide a closed loop measurement environment via temporal and energetic standardization of UV-irradiation across different skin types.
The PI-MED Procedure. Participants wore a six-aperture dose testing patch (purchased from The Daavlin Company, 2020) to allow different dosages of UV exposure across six sites on the skin. Aperture coverings were removed at timed intervals according to the published dosage schedule (Richey, et al., 2019). As UV-exposure is temporally reduced for each subsequently exposed site, PI-MED produces a gradated erythema response across the six exposure sites, such that the greatest erythema occurs in exposure site one and the least erythema response occurs in exposure site six. See Richey, and colleagues (2019) for additional details regarding materials and equipment.
Composite Erythema. Erythema, or skin redness was measured via instrumentation both prior to UV-exposure and at follow-up 24 to 48 hours subsequent to the PI-MED procedure. In accordance with prior work (Heckman et al., 2013), a handheld spectrophotometer (purchased from Konica Minolta, 2020) was used to assess erythema by measuring skin redness. Specifically, the a* scale reading from each exposure site was used as an objective measure of both pre-exposure skin color and variation in skin color induced by UV radiation 24 to 48 hours post-exposure. UV-induced erythema was computed by subtracting each exposure site’s pre-exposure a* scale reading from its post-exposure a* scale reading (Δa*). As detailed further in the section entitled ‘Assessment of PI-MED Reliability’, the Δa* measure was averaged across the six exposure sites to create a Composite Erythema measure at study baseline and at study end-point, and internal consistency of the Composite Erythemameasure was estimated across the six exposure sites’ Δa* measures. To examine changes in Composite Erythema across the study, Studywise Composite Erythema was computed by subtracting Baseline Composite Erythema from End-Point Composite Erythema.
Characterizing Skin Pigmentation.Degree of skin pigmentation is an important determinant of UV-induced erythema (Coelho, et al., 2013; Dornelles, Goldim & Cestari, 2004). The Fitzpatrick Skin Type schema (Fitzpatrick, 1988) has been used extensively to classify human skin coloration on a scale of 0—40 points, which map onto six categories of skin pigmentation from lightest (I) to darkest (VI). In the context of MED testing, this schema has been used to determine the duration of UV exposure required to produce a minimal erythema response in an individual, based on their skin type (e.g., Heckman, et al., 2013). Broadly based on the Fitzpatrick schema, the skin-typing system developed by the National Tanning Training Institute (NTTI, 2004) classifies skin coloration on a continuous scale of 0—86 points (NTTI Skin Spectrum), which map onto six primary skin type categories (NTTI Skin Type: I—VI). Previous research has validated NTTI schema (Miller, et al., 2012), and compared to the Fitzpatrick system, the NTTI system offers a more fine-grained scale, assessing skin type based on: the color of untanned skin, hair and eye color, number of freckles, ethnic background, and sunburn and tan potential.
Calibration of PI-MED to Skin Type. The current study used the six primary NTTI Skin Type categories to determine UV dosage administration in accordance with the procedures and dosage schedule developed by Richey and colleagues (2019). Following recommendations of the NTTI instrument (NTTI, 2004) and protocols reported by Richey and colleagues (2019), PI-MED testing was not conducted on individuals with Type I skin, due to the potential for UV over-dosage. Calibrating the PI-MED dosage schedule to skin pigmentation based on categorical NTTI Skin Type (II-VI) is intended to attenuate differences in erythema response that would otherwise occur if different skin types were exposed to the same amount of UV.
Accounting for Variation in PI-MED Erythema Not Fully Standardized by Calibration to Skin Type. Although the PI-MED dosage schedule is calibrated to categorical NTTI Skin Type, there remains considerable between-individual variation in erythema response, possibly due to variation in the continuous NTTI Skin Spectrum score that is collapsed when using the categorical NTTI Skin Type. In other words, the continuous NTTI Skin Spectrum variable should more precisely approximate the skin’s intrinsic sensitivity to UV radiation beyond what is accounted for by the PI-MED dosage schedule. To assess the degree to which the continuous NTTI Skin Spectrum score affected PI-MED erythema response despite the calibration for categorical NTTI Skin Type (II-VI), the continuous 0—86 NTTI Skin Spectrum score was used as an additional covariate in analyses assessing associations between PI-MED erythema and other known inflammation covariates.
Self-Report Measures of Affect
Self-report measures of affect included: (A) two anhedonia measures: Snaith–Hamilton Pleasure Scale (SHAPS; Snaith, et al., 1995) and the Dimensional Anhedonia Rating Scale (DARS; Rizvi et al., 2015), which includes an overall anhedonia measure as well as a social anhedonia subscale; (B) measures of positive and negative affect from the Positive and Negative Affect Schedule (PANAS; Watson & Clark, 1999); (C) the Perceived Stress Scale (PSS; Cohen, 1988) and (D) the Beck Depression Inventory (BDI-II; Beck, et al., 1996). All measures and relevant subscales along with sample-specific internal consistency measures are detailed in the Supplemental Method.
Several non-affective measures collected at baseline previously associated with other measures of inflammation were also examined as covariates of PI-MED erythema including age, sex, minority status, and body mass index (for review of inflammation covariates see O’Connor, 2009). It should be noted that previous research has not found age to be associated with MED and has inconsistently found MED to vary by sex (Gambichler, et al., 2006; Tan, et al., 2020). Body mass index (BMI) was only collected for 77% of the sample (n = 47) at baseline, and analyses are presented both with and without this covariate to preserve power. As previously noted, we used the continuous NTTI Skin Spectrum score as a covariate to capture additional variance in erythema response beyond that induced by PI-MED dosage based on categorical Skin Type.
To examine preliminary validation of PI-MED testing for psychoneuroimmunology research, we examined (1) the standardization of PI-MED Composite Erythema across skin pigmentation types and the reliability of the PI-MED Composite Erythema measure and (2) associations between Composite Erythema and non-affective and affective covariates.
Assessment of PI-MED Composite Erythema Standardization and Reliability. As the specific dosage schedule implemented within PI-MED is novel to MED testing (Richey, et al., 2019) and was intended to standardize erythema response across different skin pigmentation types, we (1a) used examined Composite Erythema across NTTI Skin Type using an ANOVA and follow-up t-tests, with the expectation that successful standardization of PI-MED erythema response would result in attenuated differences (ideally no differences) in Composite Erythema between the skin types. As there were only three subjects in the NTTI Skin Type IV group, this skin type was excluded from statistical analyses. Reliability of the PI-MED erythema measure was assessed by examining (1b) internal consistency across the six exposure sites (that were averaged to create the Composite Erythema measure), both at baseline and at study end-point. Specifically, Chronbach’s alpha was computed across the six Δa* values (spectrophotometer measures of pre-to-post UV exposure change in skin redness). PI-MED erythema reliability was also assessed in terms of (1c) the test-retest reliability of the Composite Erythema measures collected at baseline and at study end-point, approximately 8-10 weeks later. The test-retest reliability of Composite Erythema was examined in the whole sample, collapsing across the mindfulness and waitlist groups. Additionally, Composite Erythema test-retest reliability was also examined separately within the mindfulness and waitlist groups, as mindfulness interventions have previously been shown to lower inflammation (Dutcher, et al., 2021; Sanada, et al., 2020; Villalba, et al., 2019), suggesting that participation in the mindfulness intervention may alter test-retest associations.
Affective Correlates of PI-MED Composite Erythema. As preliminary validation of the relevance of PI-MED testing for psychoneuroimmunology research, we examined zero-order correlations between Composite Erythema and self-report measures of affect previously associated with peripheral inflammation markers, including measures of anhedonia (detailed below), PANAS Positive and Negative Affect, the Perceived Stress Scale, and the Beck Depression Inventory. Given research suggesting that inflammation may be particularly associated with symptoms of anhedonia, we hypothesized that baseline anhedonia measures, specifically, SHAPS Anhedonia, DARS Overall Anhedonia, and DARS Social Anhedonia, would show associations with Composite Erythema, beyond the effects of non-affective covariates. To test this, we estimated hierarchical regressions in which different anhedonia measures were (each separately) added to base prediction models with non-affective covariates that included NTTI Skin Spectrum, Sex, Age, Minority Status, and Body Mass Index.