Effects of Occlusion on the Skin of Atopic Dermatitis Patients

Kristen Kobaly; Ally-Khan Somani; Thomas McCormick; Susan T. Nedorost

Disclosures

Dermatitis. 2010;21(5):255-261. 

In This Article

Patients and Methods

Messenger ribonucleic acid (mRNA) produced by the skin was measured with quantitative real-time polymerase chain reaction (PCR) after a short period (6 hours) and a prolonged period (4 days) of patients' wearing the occlusive wrap and sodium lauryl sulfate (SLS). No inflammatory cytokines were found at 6 to 8 hours, so the 6-hour measurement was omitted for the last three subjects, in which inflammatory responses were measured only at the prolonged period (4 days). Changes of mRNA in interleukin-1 alpha (IL-1α), interleukin-1 receptor antagonist (IL-1RA), interleukin-8 (IL-8), and the housekeeping gene eukaryotic 18S rRNA were measured. mRNA markers were cytokines found to play a role in inflammatory response in the skin either through exacerbating (IL-1α, IL-8) or inhibiting (IL- 1RA) inflammation.[2] These inflammatory markers have been shown to be induced by SLS, our positive control.[3] 18S rRNA was used as a housekeeping gene because the expression of ribosomal RNA has been shown to be more stable than other commonly used housekeeping genes.[4–8]

Although biopsy is commonly used for the clinical investigation of the skin, this invasive method could have caused discomfort for patients participating in the study. To minimize patients' discomfort, we opted to obtain RNA for our study by using tape stripping, a technique previously shown to successfully isolate RNA from skin samples.[9–12] Benson and colleagues showed that large quantities of total RNA could be recovered from psoriatic skin via tape stripping and that tape stripping was sensitive enough to reveal mRNA markers that were undetected in biopsy specimens.[12]

Clinical Protocol

Six subjects with a history of atopic dermatitis (AD) were included. To establish the diagnosis, subjects were required to meet two of three modified Hanifin and Rajka criteria:[13] (1) chronic or relapsing eczema, (2) chronic itching affecting flexural surfaces in adults and affecting the face and extensors in infants, and (3) a personal or family history of hay fever or asthma. Exclusion criteria included the use of any topical medications on the areas of skin to be studied for 2 weeks prior to the study, current use of systemic immunosuppressants, significant exposure to natural sunlight (such as outdoor tanning) or tanning salons for 1 week prior to the study, and pregnancy. The study was approved by the University Hospitals of Cleveland Case Medical Center Institutional Review Board (#10-07-11), and informed consent was obtained from all participants.

Subjects removed heavy garments and equilibrated at room temperature for 30 minutes before the start of the study. For all subjects, the room humidity was 22.1 to 22.3%, and the room temperature was 26u to 27.7°C.

The skin of the back adjacent to the posterior axillary lines was cleaned with an alcohol pad, and a 4 × 4 cm piece of occlusive polyethylene wrap (SC Johnson Company, Racine, WI) was applied to symmetrically located sites bilaterally. Inferior to the occlusive site, a cluster of three 11 mm patch-test chambers with filters (Epitest Ltd Oy, Tuusula, Finland) each containing 30 μL of 1% SLS (Sigma-Aldrich, St. Louis, MO) was applied bilaterally. A waterproof dressing (Medline Industries Inc., Mundelein, IL) was applied over the occlusive and SLS sites to help secure the materials.

The second patient visit (subjects 1, 2, and 3 only) was 6 to 8 hours after patch application. The occlusive wrap and SLS sites were tape-stripped with D-squame adhesive 22 mm sampling discs (CuDerm Corporation, Dallas, TX), which were applied, rubbed vigorously for 10 seconds, and then removed. Twenty adhesive discs were used per site. SLS sites were tape-stripped 30 minutes after patch removal because SLS sites gradually develop more inflammation after patch tests are removed.[14] Additional occlusive dressings and SLS patch-test chambers were applied adjacent to the tape-stripped sites, to be measured at 4 days.

The third patient visit took place after 4 days, and tape stripping was done in the same way it was done at the second visit. All six subjects participated in the study during the winter months of December through February. None of the subjects had active dermatitis on the skin of the back. Consistent areas of skin were sampled for each material and each subject.

RNA Isolation

Twenty tape strips were obtained for each subject at every sampling site. The first 10 tapes from each site were discarded because we demonstrated via real-time PCR that mRNA was more abundant in the last 10 tapes (data not shown). The remaining 10 tapes were collected individually in 2 mL cryogenic vials, with the adhesive side facing inward, and stored at 280°C until processed. We added 1.5 mL of Buffer RLT (Qiagen, Valencia, CA) to the first cryogenic vial from each site. The vial was then inserted into a Braun Mikro Dismembrator (B. Braun Biotech Inc., Allentown, PA) and shaken vigorously for 2 minutes at 2,000 rpm. The vial was spun in a centrifuge at 3,000 rpm for 3 minutes at 20°C. The RLT buffer was then transferred to the next cryogenic vial in the tape series, and the process was repeated until all 10 tapes from a site were processed in the same RLT buffer. After all tapes were processed, RNA isolation was accomplished with an RNeasy Kit (Qiagen) according to the manufacturer's instructions.

Reverse Transcription and Quantitative Real-time PCR

RNA samples were reverse-transcribed into complementary deoxyribonucleic acid (cDNA) with random hexamers using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) for real-time PCR (RT-PCR). Eight microliters of RNA template were used in each cDNA reaction, in a final volume of 21μL. RT-PCR using a TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) was performed with 1.5 μL of cDNA per reaction. All reactions were run in triplicate with nontemplate controls. The specific inventoried TaqMan gene expression assays (Applied Biosystems) used included eukaryotic 18S rRNA (Hs99999901_s1), IL-8 (Hs00174103_m1), IL-1α (Hs00174092_m1), and IL-1RA (Hs00277299_m1). The mRNA levels of the targets were measured on the ABI Prism 7700 Sequence Detection System (Applied Biosystems). PCR was performed under the following conditions: 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Dilution experiments were performed to ensure similar efficiency of the PCR primer/probe sets.

PCR data are reported as the cycle threshold (Ct) necessary to achieve fluorescence. Ct values of 37 or greater were considered to have mRNA that was undetectable because Ct values beyond 37 become highly variable or fluorescence does not achieve a threshold value.[10] The relative expression levels of each gene were calculated according to the comparative, or ΔΔCt, method.[15–17] The Ct values of triplicate RT-PCR reactions were averaged for each gene in each cDNA sample. For each sample assayed, the average fluorescent reporter dye FAM (6-carboxy-fluorescein) Ct value for the gene of interest was subtracted from the average FAM Ct value of 18S rRNA to obtain the ΔCt value. The ΔΔCt value was calculated by subtracting the average ΔCt (calibrator) values from the ΔCt (sample). The relative quantification was calculated by 2−ΔΔCt. The mRNA quantity for the calibrator is expressed as 1 × sample, and all other quantities are expressed as a fold difference relative to the calibrator. The mean ΔCt of the SLS was used as the calibrator in all tests. The standard deviation values for ΔΔCt were added to or subtracted from the ΔΔCt to generate the upper and lower ΔΔCt standard deviation boundaries. These ΔΔCt values were then transformed (2−ΔΔCt ±s, where "s" equals the standard deviation of the ΔΔCt value) to generate error bars. In all statistical analyses, significance was assessed by means of Student's t-test, and p < 0.05 was considered significant.

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