Written, informed consent was obtained from all participants before enrollment, and all clinical investigations were conducted according to the Declaration of Helsinki principles. The research protocol was approved by the human studies committees at the University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, University of Osnabruck, FRG, and the National Hospital of Colombo, Sri Lanka.
Skin surface pH was measured in 110 healthy nurses (72 females, mean age 29 ± SD 6.6) with type I–II skin (Fitzpatrick scale) working at the University of Osnabruck, Osnabruck, Germany, and in 129 nurses (117 females, mean age 25 ± SD 2.1 years) with type IV–V skin, working at the National Hospital, Colombo, Sri Lanka. Epidermal integrity was assessed in two groups of healthy volunteers, each having 20 subjects with type I–II (age 31.8 ± SD 6.9 years) and type IV–V (age 32.4 ± SD 9.7 years) skin.
To ascertain if the results from the functional studies are reproducible in subjects of pigment extremes from the same geographic location, functional measurements were repeated in 17 healthy volunteers (10 subjects with type I–II and 7 subjects with type IV–V skin pigment types) at the Veteran Affairs Medical Hospital, San Francisco, CA. Normal human skin was obtained for morphological and tissue culture studies from fresh surgically resected “dog ears,” in compliance with Declaration of Helsinki principles.
Volunteers refrained from using skin-cleansing agents or other local applications for at least 48 hours before and during the study. Subjects with current or previous skin disease were excluded. After a 15-minute acclimatization period, functional measurements were taken in a controlled environment with temperature and relative humidity set between 22 and 25 °C and between 40 and 60%, respectively. A flat glass electrode (Mettler-Toledo, Giessen, Germany), attached to a precision pH meter (pH 900; Courage & Khazaka, Cologne, Germany), was used to measure skin surface pH on the volar forearm and the dorsa of the nondominant hands of the volunteers.
To assess SC integrity and permeability barrier recovery, TEWL was first measured under basal conditions, on a circular area of 1 cm diameter, on the volar aspect of the nondominant forearm, using a Tewameter (TM 300; Courage & Khazaka) following the guidelines (Pinnagoda et al., 1990
). TEWL values were registered in gm−2
after equilibration of the probe on the skin. Sequentially, 3M Blenderm tape strips (3M Health Care, Neuss, Germany) were pressed, with comparable pressure to the test sites for about 3 seconds each, and removed with forceps. TEWL levels were measured after every five tape strippings. Tape stripping was repeated until TEWL increased by threefold. Barrier recovery was evaluated at 24, 48, and 72 hours after tape stripping. For calculation of the percentage change in TEWL, the following formula was used: (TEWL immediately after stripping−TEWL at the indicated time)/(TEWL immediately after stripping−baseline TEWL) × 100%.
To assess SC cohesion, sequential D-squame tapes were applied on the volar forearm of healthy volunteers as described above, and removed until TEWL is increased by threefold. Amount of protein removed per tape was estimated using Bio-Rad protein assay, as described previously (Dreher et al., 1998
Sixteen normal human volunteers (four males and twelve females; ages 32 ± 9 years) were included after providing informed consent. To modulate the pH sustainably on the forearms of human volunteers, we applied 500 μl of (1) LBA and NaOH-neutralized LBA (5% vol/vol in propylene glycol/ethanol, 7:3, pH 3.2), (2) GL and NaOH-neutralized GL, or (3) vehicle, without occlusion, randomly to contralateral forearms. Surface pH was measured at 1, 6 and 24 hours after LBA/GL or vehicle application.
To assess permeability barrier function, TEWL was measured first under basal conditions, as well as immediately following acute barrier disruption by repeated D-squame tape stripping (20–25 Dsquame tapes increased EWL to ≥20 mgcm−2 hour−1), and 3, 24, and 48 hours after application of LBA/neutralized LBA, GL/neutralized GL, and propylene glycol/ethanol vehicle. The area under the curve was calculated.
Ultrastructural and quantitative morphological studies
Biopsy samples were minced to < 0.5mm3, fixed in modified Karnovsky’s fixative (2% paraformaldehyde, 2% glutaraldehyde, 0.1M cacodylate buffer, pH 7.4) overnight, and postfixed with either 0.25% ruthenium tetroxide or reduced osmium (1% aqueous osmium tetroxide, 1.5% potassium ferrocyanide). After postfixation, samples were dehydrated in a graded ethanol series, and embedded in an Epon-epoxy mixture. Ultrathin sections were cut on an ultramicrotome (Leica Ultracut E, Nussloch, Germany) and examined in an electron microscope (Zeiss 10A; Carl Zeiss, Thornwood, NY) operated at 60 kV. At least 10 random images from each subject (n = 5 from each pigment group) were taken at × 25 by an unbiased observer and used for quantitative assessments.
The ratio between total length of intact CDs to total length of cornified envelopes was determined in the first two layers above the SC–SG junction, and in the 2–3 outermost layers of the SC by planimetry (n = 5 subjects from each pigment group).
Lamellar body density was measured in the two SG layers, immediately beneath the SC–SG junction by randomly superimposing a stereological grid and counting “hits” versus “non-hits.” LB density was expressed as hits/(hits + nonhits) × 100 (n = 5 subjects from each pigment group).
The quality and quantity of the lamellar bilayers in the SC was assessed in randomized, coded micrographs after ruthenium tetroxide postfixation by a blinded observer (P.M.E.).
Changes in CE thickness of corneocytes in the outer and lower SC in each subject were measured in randomized, coded electron micrographs using Gattan software. At least 30 measurements were taken from at least 5 subjects in each pigment group.
The number of cell layers in the SC was counted in at least two low-power (× 3,000) electron micrographs from each subject (n = 5 for each pigment group).
In situ zymographic assays
Surgical biopsies (n
= 4 from each pigment group) were snap-frozen and stored at −80 °C. Frozen sections (7 μm) were rinsed with washing solution (0.025% Tween-20 in deionized water) and incubated at 37 °C for 2 hours with 250 μl BODIPY-Fl-casein (1 μg μl−1
) in deionized water (3 μlml−1
). Some sections were exposed to the fluorophore substrate in a neutral buffer (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4). Sections were then rinsed with 0.025% Tween-20 washing solution, coverslipped, counterstained with propidium iodide (Sigma Aldrich, Bornem, Belgium), and visualized under a confocal microscope (Leica TCS SP, Heidelberg Germany) at an excitation length of 485nm and emission wavelength of 530 nm. Zymographic assays of enzyme activity, although not quantitative, show in situ
activity more accurately than in vitro
assays, where the pH of the buffer solution artificially changes activities (for example, Hachem et al., 2003
Frozen sections (7 μm) were rinsed with washing solution (0.025% Tween-20 in deionized water) and incubated at 37 °C for 2 hours with 250 μl BODIPY-Fl-pepstatin A (1 μg μl−1) in deionized water (1 μlml−1). Sections were then rinsed with 0.025% Tween-20 washing solution, coverslipped, counterstained with propidium iodide (Sigma Aldrich), and visualized under a confocal microscope (Leica TCS SP) at an excitation length of 485nm and emission wavelength of 530 nm.
Immunohistochemistry and immunofluorescence
Immunohistochemical staining for assessment of changes in epidermal differentiation was performed as described earlier (Demerjian et al., 2006
). Briefly, after deparaffinization, 5 μm sections were incubated with the primary antibodies overnight at 4 °C. After washes ×3, sections were incubated with the secondary antibody for 30 minutes. Staining was detected with ABC-peroxidase kit obtained from Vector Labs (Burlingame, CA). After counter-staining with hematoxylin, sections were visualized under a light microscope, and digital images were captured with AxioVision software (Carl Zeiss Vision, Munich, Germany).
Immunofluorescence was used to detect DSG-1. After deparaffinization, 5 μm paraffin sections were rehydrated with distilled water, washed with 1 ×TBS, incubated for 30 minutes in blocking buffer (1% bovine serum albumin, 0.1% cold-water fish gelatin in phosphate buffered saline), and were then incubated overnight at 4 °C with mouse anti-human DSG-1 monoclonal antibody (Millipore, Billerica, MA) in blocking buffer. Tissue sections were then washed with 1 × TBS, and incubated for 1 hour with Alexa Fluor 488 secondary antibody in blocking buffer, counterstained with propidium iodide (Sigma Aldrich), and visualized in a confocal microscope (Leica TCS SP) at an excitation length of 485nm and emission wavelength of 530 nm.
Lipid and melanin detection
Frozen sections (7 μm) were incubated with Nile Red (Sigma Aldrich) in 75% glycerol (2 μgml−1) for 5 min and visualized in a confocal microscope (Leica TCS SP) at an excitation length of 485nm and emission wavelength of 530 nm.
Fontana–Masson stain for melanin detection
After deparaffinization, 5 μm sections were incubated with fresh ammoniacal silver solution in a 55 °C water bath for 30 minutes. Slides were then placed in 0.1% gold chloride for 1 minute and in 5% sodium thiosulphate for 2 minutes, counterstained with Nuclear Red fast, and visualized under a light microscope. Digital images were captured with AxioVision software (Carl Zeiss Vision).
Two-channel confocal imaging of cultured keratinocytes
Human melanocytes from darkly and lightly pigmented neonatal foreskin samples (n > 3 each) were plated in separate four-well coverslips in a melanocyte growth medium (Cascade M254-500). Human keratinocytes from neonatal foreskin samples were plated in two-well coverslips in C-154 media with KGS.
Immediately before incubation, a 10 μM SNARF-5F-AM (Molecular Probes, Eugene, OR) solution in melanocyte growth medium was prepared. Cells were incubated with the 10 μM dye solution for about 1 hour at 5% CO2 and 37 °C. Before imaging, the dye containing medium was removed and the cells were rinsed once with melanocyte-growing media. A Zeiss LSM 510 Meta (Zeiss, Jena, Germany) was used to detect the pH-dependent spectral changes of the SNARF emission. The 488nm line of the Argon laser was used as the excitation line. A dichroic mirror reflecting wavelengths shorter than 635nm was used to split the fluorescence emission between two emission channels (Ch1 and Ch2). The Meta detector (used as Ch1) was set to detect light with wavelength longer than 623 nm. A band-pass filter centered at 563nm with a width of 55nm was placed in front of the PMT in Ch2. The pinholes in front of Ch1 and Ch2 were adjusted to give an optical slice of 0.8 μm with a ×63 oil objective. The gain and offset levels of the detectors were independently adjusted to ensure sensitivity in the pH range from 6 to 8. This was done by imaging a 5 μM solution of SNARF-5F in phosphate buffers at pH 5, 6, 7.4, and 8. The offset and gain levels of the detectors were then kept constant for all of the experiments.
Fluorescence intensity images collected in each channel were processed using Matlab (MathWorks, Natick, MA) after first eliminating artifacts due to a nonhomogeneous fluorescence intensity. Ch1 intensity was used to ascertain threshold levels of fluorescence for both channel intensity images. The spectral changes in the SNARF-5F emission were quantified by calculating the quantity R
, defined below, pixel by pixel from the two images using the formula:
where Ich1 and Ich2 are the intensities in Ch1 and Ch2, respectively. region of interests (ROIs) corresponding to cell bodies or dendrites were selected on the normalized ratio image using Image J software, and the average R
values and SD are calculated for each ROI.
The values of R were converted to pH by performing an in-cell calibration in human keratinocytes. A pH 7.8 buffer with 13.5 μM nigericin (Sigma Aldrich) as the permeabilizing agent was added to one of the two wells, and keratinocytes were imaged every 10 seconds for about 20 minutes (we determined that about 10 minutes are needed for the intracellular pH spectra to equilibrate with the extracellular buffer). The same procedure was repeated on the second well of the cover slip using a 6.5 pH buffer with 13.5 μM nigericin. A linear dependence was assumed between R and pH. This assumption is based on the observed linear dependence of R on pH in solution (data not shown) over a pH range from 6 to 8.
Organotypic cell cultures
Primary cultures of human keratinocytes and melanocytes were established from neonatal foreskins of designated pigment types. Human keratinocytes and melanocytes were pipetted into polyethylene-coated transwells containing CnT-07 medium (Progenitor Cell Targeted culture medium with 0.07mM Ca+ +; CELLnTEC Advanced Cell Systems, Bern, Switzerland) at 6:1 ratio, and incubated at 37 °C and 5% CO2 for 72 hours. Cultures were then switched to CnT-02 medium (differentiation medium containing 1.2mM Ca+ +) and exposed to air–liquid interface after 16 hours by removing most of the medium from transwells. They were then maintained at 37 °C with frequent media changes and harvested on day 10.
Two groups were compared with a Student’s t-test. Nonparametric Mann–Whitney statistical analyses were performed to compare percentage of ratios between different groups of treatments. Statistical analyses were performed using Prism 3 (GraphPad software, San Diego, CA).