BJD

C L I N I C A L A N D L A B O R A T O R Y I N V E S TI G A T I O N S British Journal of Dermatology

Inuence of Aqueous Cream BP on corneocyte size, maturity, skin protease activity, protein content and transepidermal water loss
D. Mohammed, P.J. Matts,* J. Hadgraft and M.E. Lane
Department of Pharmaceutics, School of Pharmacy, 2939 Brunswick Square, London WC1N 1AX, U.K. *Procter & Gamble Technical Centres Ltd, London Innovation Centre, Whitehall Lane, Egham, Surrey TW20 9NW, U.K.

Summary
Correspondence
Majella E. Lane. E-mail: majella.lane@btinternet.com

Accepted for publication

21 February 2011

Funding sources
No external funding.

Conicts of interest
None declared. DOI 10.1111/j.1365-2133.2011.10338.x

Background Aqueous Cream BP is frequently prescribed for patients with eczema and is known to induce sensitivity in certain patients and also to decrease the thickness of the stratum corneum (SC). We have previously reported methodology to quantify corneocyte maturity and size, protease activity and protein content within different levels of the SC. Objectives The aim of the present study was to investigate changes in corneocyte size, corneocyte maturity, selected protease activities, protein content and transepidermal water loss (TEWL) in normal skin after a 28-day application of Aqueous Cream BP. Methods The left and right mid volar forearms of six healthy female volunteers were selected as the study sites. Aqueous Cream BP was applied twice daily to treated sites for 28 days. At the end of this period, the site was tape-stripped and corneocyte maturity, corneocyte size and protease activity of the desquamatory kallikrein proteases, KLK5 and KLK7, and the inammatory proteases tryptase and plasmin were measured. Protein content and TEWL measurements were also recorded. Results Corneocyte maturity and size decreased with increasing number of tape strips, and were signicantly lower in treated sites compared with untreated sites. Protease activity and TEWL values were higher (P < 005) for the treated sites compared with untreated sites. The amount of protein removed from deeper layers of treated sites was signicantly lower than from untreated sites. Conclusions We report rapid minimally invasive measures of the effects of Aqueous Cream BP at the cellular and molecular level of the skin. Treatment with this formulation is associated with increased desquamatory and inammatory protease activity. Changes in corneocyte maturity and size are also indicative of accelerated skin turnover induced by chronic application of this emollient. These ndings question rmly the routine prescription of this preparation as a moisturizer in patients with atopic dermatitis.

Eczema is reported to account for up to 225% of cutaneous diseases treated by general practitioners in the U.K.1 For the management of eczema and dry skin the British National Formulary currently includes Aqueous Cream BP under the heading of Emollients, which are further dened as preparations which soothe, smooth and hydrate the skin and are indicated for all dry or scaling disorders.2 Aqueous Cream BP rst appeared as a preparation of the British Pharmacopoeia in 1958 and its formulation, containing sodium lauryl sulphate (SLS), remains largely unchanged today.3 The continued recommendation of

Aqueous Cream BP as an emollient is surprising as SLS is a known skin irritant.47 and it is commonly used in patch testing. The use of Aqueous Cream BP is also associated with a number of adverse skin reactions in children8 and more recently with signicant thinning of the stratum corneum (SC).9 The exact mechanism by which the components of Aqueous Cream BP contribute to these effects is not understood. We have recently reported the development of rapid minimally invasive measures of the spatial distribution of corneocyte maturity and size as well as protease activity and protein con 2011 The Authors

1304

BJD 2011 British Association of Dermatologists 2011 164, pp13041310

Inuence of Aqueous Cream BP on skin characteristics, D. Mohammed et al. 1305

tent within different levels of the SC layers.10 Quantitative indices of the mature, rigid corneocyte envelopes isolated from the outermost layer of the SC and the fragile, less mature corneocyte envelopes present in deeper layers of the SC were reported. Corneocyte size measurements were also observed to be greater for the more mature skin cells when compared with the less mature cells deeper in the SC. The activities of the key desquamatory kallikrein enzymes, tryptic-like KLK5 and chymotryptic-like KLK7, together with inammatory-related protease tryptase were also evaluated as a function of depth in the SC. In the present study we examine the effects of the application of Aqueous Cream BP on these markers of epidermal turnover and skin barrier function. We have also extended the proteases studied to include plasmin as this enzyme appears to be the most responsive inammatory-related marker for the skin.11,12 As transepidermal water loss (TEWL) is routinely used to characterize skin barrier function TEWL measurements were also conducted.13

research protocol was approved by the South West London Research Ethics Committee (Reference 10 H0801 69). A participant information leaet was supplied to the volunteers prior to the study. Subjects were asked, apart from daily washing, not to apply any moisturizer or cosmetic product to the region of interest during the study. Application of Aqueous Cream BP Aqueous Cream BP was applied according to a previously published protocol.9 Briey, the left and right mid volar forearm were delineated to provide well-separated treatment and control sites. Cream (2 mL) was applied using a needleless syringe for 10 min to the treatment sites (approximately 40 cm2) twice a day for 28 days. Excess cream was gently removed by soft facial tissue away from the control sites. A 1-day washout period was allowed before any experimental measurements, during which participants refrained from the use of any moisturizing or other skin care treatment on their forearms. Intra individual and regional differences were minimized by conducting both control and treated experiments on the same arm. Tape stripping Standard D-Squame tape was applied to the forearm sites at a constant pressure as described previously.10 Four areas (two within the treated and two within the untreated sites) within each mid volar forearm were selected. Twenty consecutive tape strippings were performed for all the selected areas within the treated and untreated sites. Transepidermal water loss measurement Volunteers were allowed to acclimatize for 15 min in a controlled atmosphere (20 1 C and 45 5% relative humidity) prior to the tests, in order to equilibrate to room conditions and to minimize insensible perspiration. TEWL was then measured (using an Aquaux AF102 instrument; Biox Systems Ltd). TEWL measurements were performed initially and after each series of four tape strips were removed. Protein content Protein content (Cprotein) was measured directly from the tape strips. Protein absorption was determined at 850 nm using an infrared densitometer SquameScan 850A (Heiland Electronic) as previously reported.11 The amount of protein was subsequently quantied as follows:11 Cprotein lg cm2 1366 Absorption % 1557

Materials and methods

Materials Standard D-Squame tape (22 cm in diameter, 38 cm2) was obtained from CuDerm Corporation (Dallas, TX, U.S.A). Sodium lauryl sulphate, ethylenediaminetetraacetic acid, Triton X-100, acetic acid and Nile red were obtained from SigmaAldrich (Gillingham, U.K.). HPLC analytical grade water and methanol were obtained from Fisher Scientic (Loughborough, U.K.). DL-Dithiothreitol and TrisHCl buffer (pH 80) were obtained from Fluka Analytical (Gillingham, U.K). Dimethyl sulphoxide was obtained from VWR International Ltd (Lutterworth, U.K.) and phosphate-buffered saline tablets were obtained from Oxoid (Cambridge, U.K.). TEWL was measured using an Aquaux AF103 (Biox Systems Ltd, London, U.K.) and protein absorbance was measured at 850 nm using a SquameScan A850 infrared densitometer (Heiland Electronic, Wetzlar, Germany). The primary monoclonal antibody, antihuman involucrin (clone SY5), was purchased from Cambridge Scientic, Monosan (Cambridge, U.K.) and the rabbit polyclonal antibody to mouse uorescent IgG H & L (whole molecule) uoresceinisothiocyanate antibody was obtained from Abcam (Cambridge, U.K.). Aminomethyl coumarin (AMC) and all uorogenic peptide substrates were generous gifts from Pentapharm Ltd (now part of DSM Nutritional Products Ltd, Basel, Switzerland). Aqueous Cream BP was obtained from Boots The Chemist (The Boots Company PLC, Nottingham, U.K.). Methods Volunteer recruitment Six healthy female Caucasian volunteers aged 2329 years were recruited in November 2010 and the study was performed from mid November 2010 to mid December 2010. None of the volunteers had any history of skin disease. The
2011 The Authors BJD 2011 British Association of Dermatologists 2011 164, pp13041310

Corneocyte maturity Corneocyte maturity was measured by immunouorescence and Nile Red staining of tape strip numbers 1, 5, 9, 13 and 17 and full details of the methodology have been reported elsewhere.10

1306 Inuence of Aqueous Cream BP on skin characteristics, D. Mohammed et al.

TEWL (g m2 h1)

Fluorescence was monitored with a uorescence microscope equipped with a Fuji S2 Pro 6.2 megapixel camera (Fuji, Tokyo, Japan). The object was magnied 10, giving 08 pixels lm)1 with a resolution of 1440 960 pixels. ImageJ image analysis software (NIH-Image, Bethesda, MD, U.S.A.) was used to analyse the red pixels obtained from Nile Red stained cells and the green pixels from the immunostained cells. Measurement of corneocyte surface area ImageJ imaging software was calibrated to measure the corrected number of pixels based on the images acquired (08 pixels lm)1 with a resolution of 1440 960 pixels).10 Corneocyte surface areas were measured for the same tape strip numbers as those measured for corneocyte maturity, i.e. tape numbers 1, 5, 9, 13 and 17. A minimum of 10 images, randomly selected, was taken from each slide. Protease activity Stratum corneum samples were obtained by tape stripping as described above. Protease activity was measured for tape strip numbers 24, 68, 1012, 1416 and 1820 as previously described.10 Statistical analysis SPSS version 18 (SPSS, IBM, Somers, NY, U.S.A.) was used to analyse the data. Parametric statistical tests (one-way betweengroup ANOVA and paired t-test to compare means) were used to investigate statistical differences between treated and untreated sites. A probability of P < 005 was considered statistically signicant. All results are presented as mean SEM.

45 40 35 30 25 20 15 10 5 0 14 58

Control sites Treated sites

912

1316

1720

Tape strips

Fig 1. Transepidermal water loss (TEWL) measurement of the mid volar forearm with increasing number of tape strips for control and treated sites (n = 24, mean SEM).

25

Control sites Treated sites

Protein content (g cm2)

20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Number of tape strips

Fig 2. Stratum corneum protein content removed from mid volar forearm for each tape strip for control and treated sites (n = 24, mean SEM).

Results
TEWL measurements for control and treated sites are shown in Figure 1. TEWL measurements were conducted before tape stripping and after removal of 4, 8, 12, 16 and 20 tape strips. The difference between control and treated sites was statistically signicant up to removal of 16 tape strips (P < 005), i.e. with increasing amounts of stratum corneum removed, water loss was always higher for sites exposed to Aqueous Cream BP compared with untreated sites. After removal of 1720 tape strips, the mean value of TEWL was not signicantly lower for treated sites vs. control sites (P = 009). The initial values of TEWL were of a similar magnitude to those reported by Tsang and Guy9 for control sites and sites treated with Aqueous Cream BP over the same time-frame and using the same instrumentation. Protein content Figure 2 shows the protein content, which was measured for each individual tape strip. As noted previously10 the amount of protein removed decreased was more SC was removed, reecting increased cohesion of the deeper layers.1416

Signicant differences (P < 005) in protein content between control and treated sites were evident after removal of 12 tape strips. A correlation between amount of protein removed and weight of tape strips also allowed an estimation of the depth of SC probed for both control and treated sites (data not shown). For the same number of tape strips, the depth to which the SC was tape stripped may be estimated as 826 150 lm for control sites and 758 109 lm for treated sites (P < 005). Corneocyte maturity Representative micrographs showing corneocytes stained with Nile Red and immunostained for native involucrin to assess corneocyte maturity are shown in Figure 3. More mature corneocytes can be seen in control-site micrographs when compared with those for the treated site. Figure 4 illustrates the maturity of corneocytes for both control and treated sites (calculated as a ratio of red green pixels, described previously10). For the control site, more mature cells were found in the outer layers of the SC with progressively fewer mature corneocytes found with increasing depth into the SC.
2011 The Authors BJD 2011 British Association of Dermatologists 2011 164, pp13041310

Inuence of Aqueous Cream BP on skin characteristics, D. Mohammed et al. 1307

(a)

(c)

100 m

100 m

(b)

(d)

100 m

100 m

Fig 3. Micrographs showing corneocyte maturity from tape 1 of the volar forearm of a subject. (a, c) are control and treated, respectively, stained with Nile Red. (b, d) are control and treated, respectively, and immunostained for native involucrin; 1440 960 pixels, 08 pixels lm)1.

There were signicant differences (P < 005) in the maturity of corneocytes from control sites when compared with treated sites. For all series of tape strips analysed, corneocytes were always more mature in control sites than in treated sites. Corneocyte surface area Corneocyte surface area decreased with each tape strip series for both groups (Fig. 5). However, the cells from control sites were signicantly larger (P < 005) than those from the treated sites, consistent with the maturity data above (Fig. 4). When the correlation between corneocyte maturity and surface area was probed using simple regression analysis for both treated and control groups, high correlation coefcients were obtained (i.e. more mature corneocytes were larger, in line with our previous ndings10), providing further condence in

the robustness of the methods used to determine these properties (Fig. 6). Protease activity For the proteases studied (KLK5, KLK7, tryptase and plasmin), activity was found to decrease with increasing depth (Fig. 7). For both control and treated sites, elevated activities of the desquamatory and inammatory enzymes were observed in the outermost layers of the SC compared with the deeper layers, in line with the ndings of Voegeli et al.11 and with our own work.10 There were signicant differences in the activities of all proteases for control vs. treated sites (P < 005). In all cases, treatment with Aqueous Cream BP resulted in signicantly increased activity of the desquamatory kallikreins as well as the

Corneocyte maturity (RED/GREEN pixel ratio)

6 5 4 3 2 1 0 1 5 9 13

Control sites

105000

Corneocyte surface area (m2)

Treated sites

Control sites Treated sites

100000 95000 90000 85000 80000 75000 70000 65000

17

13

17

Number of tape strips

Number of tape strips

Fig 4. Corneocyte maturity of skin samples removed from the mid volar forearm by tape stripping (n = 24, mean SEM). 2011 The Authors BJD 2011 British Association of Dermatologists 2011 164, pp13041310

Fig 5. Mean corneocyte surface area of skin samples removed from the mid volar forearm by tape stripping (n = 24, mean SEM).

1308 Inuence of Aqueous Cream BP on skin characteristics, D. Mohammed et al.

105000 Control sites

Corneocyte surface area (m2)

100000 95000 90000 85000 80000 75000 70000 0

Treated sites

Corneocyte maturity (RED/GREEN pixel ratio)

Fig 6. Correlation of corneocyte maturity with corneocyte size for control and treated sites. Control ( ) y = 557x + 7614, r2 = 091; treated ( ) y = 842x + 6936, r2 = 083 (n = 24; mean SEM).

inammatory enzymes, plasmin and tryptase at all SC depths assayed. The ndings are also consistent with those of Suzuki et al.,17,18 where treatment with SLS resulted in an increase in trypsin-like and chymotrypsin-like protease activity in the SC.

Discussion
For effective turnover of the skin, proteolysis of the cohesive corneodesmosomes which bind the corneocytes in the SC

must occur. This process is mediated by proteases which catalyse peptide bond hydrolysis.19 The human tissue kallikreins (KLKs) are found in a variety of tissues20 and have been suggested to function as an enzymatic cascade pathway.2123 Corneodesmosomes are known to be broken down by several of the desquamatory-related serine proteases, such as KLK5, KLK6, KLK7, KLK8, KLK10, KLK11 and KLK13, present within the SC.17,24 As well as having a vital role in the proteolysis of the SC corneodesmosomes, KLKs are also reported to be involved in degradation of lipid-processing enzymes, such as b-glucocerebrosidase.25 The expression of tryptic (e.g. KLK5) and chymotryptic (e.g. KLK7) enzymes has been reported to be reduced in the outer layers of the SC in dry skin.2628 However, increased expression of KLK7 has been reported in two major chronic inammatory diseases: psoriasis and atopic dermatitis.24,29 Changes in the activity of serine proteases KLK5 and KLK7 also appear to be associated with skin barrier disturbances.3032 This may partly be due to uncontrolled corneodesmolysis leading to weakening of SC integrity and cohesion.25,27 Other enzymes such as plasmin, tryptase and urokinase are also reported to be present in the SC but are not necessarily involved in the desquamatory process. However, these enzymes may have an important role in the inammatory process in the skin.11,29 Because chronic use of Aqueous Cream BP has previously been associated with skin irritation and thinning of the skin, we hypothesized that these effects might be linked to the

KLK 5 activity (nU g protein1)

12 10 8 6 4 2 0 24 68 1012

Control sites Treated sites

KLK 7 activity (nU g protein1)

14

(a)

16 14 12 10 8 6 4 2 0

(b)

Control sites Treated sites

1416

1820

24

68

1012

1416

1820

Pooled tape strips Tryptase activity (nU g protein1)

25 23 21 19 17 15 13 11 09 07 05 24 68 1012 1416 1820

Pooled tape strips

4 (d) 35 3 25 2 15 1 24 68 1012 1416 1820

Plasmin activity (nU g protein1)

(c)

Control sites Treated sites

Control sites Treated sites

Pooled tape strips

Pooled tape strips

Fig 7. Stratum corneum protease activity of the mid volar forearm with depth; (a) KLK5; (b) KLK7; (c) tryptase; (d) plasmin (n = 24, mean SEM). 2011 The Authors BJD 2011 British Association of Dermatologists 2011 164, pp13041310

Inuence of Aqueous Cream BP on skin characteristics, D. Mohammed et al. 1309

inuence of this formulation on the molecular markers of epidermal turnover such as protease activity, corneocyte maturity, size and skin protein content. The application of Aqueous Cream BP appears to disrupt the normal maturation process, i.e. the corneocytes do not mature fully because of exposure to the preparation. The ndings of our study also conrm increased protease activity for both the desquamatory enzymes KLK5 and KLK7, and the inammatory enzymes plasmin and tryptase with repeated application of Aqueous Cream BP. Elevated levels for the latter two enzymes also suggest an inammatory response in sites treated with this preparation and, indeed, the reduction in corneocyte surface area and maturity is entirely consistent with increased epidermal turnover. The depth to which skin could be probed for treated sites was approximately 07 lm less than for control sites. This compares well with the reported decrease in SC thickness of 11 lm, following the same treatment protocol by Tsang and Guy.9 Finally, increased TEWL values are consistent with this disturbance in corneocyte maturation during the treatment period. The results provide a mechanistic understanding for the observed thinning of the SC associated with use of Aqueous Cream BP and for the irritant effects reported following the use of this preparation in patients with atopic dermatitis. In conclusion, robust, minimally invasive and sensitive methods have, for the rst time, been developed and used to delineate the effects of Aqueous Cream BP on healthy skin at the cellular and molecular level. It is reasonable to hypothesize that, in patients with atopic dermatitis, further deterioration in these markers of skin health would be manifested with continual use of Aqueous Cream BP. Future studies will extend these ndings to evaluate the effects in a cohort of patients with a history of atopic dermatitis and to examine the effects of other dermatological preparations (and their constituents) on the SC biomarkers described in this publication.

Acknowledgment
Diar Mohammed wishes to acknowledge Proctor & Gamble for studentship funding.

References
1 Kerr OA, Tidman MJ, Walker JJ et al. The prole of dermatological problems in primary care. Clin Exp Dermatol 2009; 35:3803. 2 British National Formulary 60. London: British Medical Association and Royal Pharmaceutical Society of Great Britain, September 2010; 68990. 3 British Pharmacopoeia. London: The Pharmaceutical Press, 1958. 4 Dahl MV, Trancik RJ. Sodium lauryl sulfate irritant patch tests: degree of inammation at various times. Contact Dermatitis 1977; 3:2636. 5 Bruynzeel DP, van Ketel WG, Scheper RJ, von Blomberg-van der Flier BM. Delayed time course of irritation by sodium lauryl sulfate: observations on threshold reactions. Contact Dermatitis 1982; 8:2369. fer H, Pirker C, Aramaki J et al. Evaluation of skin susceptibility 6 Lo to irritancy by routine patch testing with sodium lauryl sulfate. Eur J Dermatol 2001; 5:41619. 7 Geier J, Uter W, Pirker C, Frosch PJ. Patch testing with the irritant sodium lauryl sulfate (SLS) is useful in interpreting weak reactions to contact allergens as allergic or irritant. Contact Dermatitis 2003; 48:99107. 8 Cork MJ, Timmins J, Holden C et al. An audit of adverse drug reactions to aqueous cream in children with atopic eczema. Br J Dermatol 2004; 151 (Suppl. 68):57. 9 Tsang M, Guy RH. Effect of Aqueous Cream BP on human stratum corneum in vivo. Br J Dermatol 2010; 163:9548. 10 Mohammed D, Matts PJ, Hadgraft J, Lane ME. Depth proling of the stratum corneum biophysical and molecular properties. Br J Dermatol 2011; 164:95765. 11 Voegeli R, Rawlings AV, Doppler S et al. Proling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase-like enzyme. Int J Cosmet Sci 2007; 29:191200. 12 Voegeli R, Rawlings AV, Doppler S, Schreier T. Increased basal transepidermal water loss leads to elevation of some but not all stratum corneum serine proteases. Int J Cosmet Sci 2008; 30:435 42. 13 Endo K, Suzuki N, Yoshida O et al. The barrier component and the driving force component of transepidermal water loss and their application to skin irritant tests. Skin Res Technol 2007; 13:42535. 14 Dreher F, Arens A, Hostynek JJ et al. Colorimetric method for quantifying human stratum corneum removed by adhesive-tape stripping. Acta Derm Venereol 1998; 78:1869. 15 Bashir SJ, Chew AL, Anigbogu A et al. Physical and physiological effects of stratum corneum tape stripping. Skin Res Technol 2001; 7:408. 16 Dreher F, Modjtahedi BS, Modjtahedi SP, Maibach HI. Quantication of stratum corneum removal by adhesive tape stripping by total protein assay in 96-well microplates. Skin Res Technol 2005; 11:97101. 17 Suzuki Y, Nomura J, Hori J et al. Detection and characterization of endogenous protease associated with desquamation of stratum corneum. Arch Dermatol Res 1993; 285:3727.

Whats already known about this topic?

The repeated application of Aqueous Cream BP is known to induce skin irritation and has been associated with a decrease in skin thickness. The exact mechanism by which the components of Aqueous Cream BP contribute to these effects is not known.

What does this study add?

Treatment with Aqueous Cream BP increases desquamatory and inammatory protease activity. Accelerated skin turnover is also induced by chronic application of this emollient. Aqueous Cream BP should not be prescribed as a moisturizer for patients with atopic dermatitis.

2011 The Authors BJD 2011 British Association of Dermatologists 2011 164, pp13041310

1310 Inuence of Aqueous Cream BP on skin characteristics, D. Mohammed et al. 18 Suzuki Y, Nomura J, Koyama J, Horii I. The role of proteases in stratum corneum: involvement in stratum corneum desquamation. Arch Dermatol Res 1994; 286:24953. 19 Rawlings N, Barrett A. MEROPS: the peptidase database. Nucleic Acids Res 1999; 27:32531. 20 Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr Rev 2001; 22:184204. 21 Yousef GM, Diamandis EP. Human tissue kallikreins: a new enzymatic cascade pathway? Biol Chem 2002; 383:104557. 22 Brattsand M, Stefansson K, Lundh C et al. A proteolytic cascade of kallikreins in the stratum corneum. J Invest Dermatol 2004; 124:198 203. 23 Borgono CA, Michael IP, Diamandis EP. Human tissue kallikreins: physiologic roles and applications in cancer. Mol Cancer Res 2004; 2:25780. 24 Komatsu N, Saijoh K, Toyama T et al. Multiple tissue kallikrein mRNA and protein expression in normal skin and skin diseases. Br J Dermatol 2005; 153:27481. 25 Hachem J-P, Man M-Q, Crumrine D et al. Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity. J Invest Dermatol 2005; 125:51020. 26 Redoules D, Tarroux R, Assalit MF, Peri JJ. Characterisation and assay of ve enzymatic activities in the stratum corneum using tape-strippings. Skin Pharmacol Appl Skin Physiol 1999; 12:18292. 27 Harding CR, Watkinson A, Rawlings AV, Scott IR. Dry skin, moisturization and corneodesmolysis. Int J Cosmet Sci 2000; 22:2152. 28 Overloop LV, Declercq L, Maes D. Visual scaliness of human skin correlates to decreased ceramide levels and decreased stratum corneum protease activity. J Invest Dermatol 2001; 117:811. 29 Voegeli R, Rawlings AV, Breternitz M et al. Increased stratum corneum serine protease activity in acute eczematous atopic skin. Br J Dermatol 2009; 161:707. 30 Egelrud T, Brattsand M, Kreutzmann P et al. hK5 and hK7, two serine proteinases abundant in human skin, are inhibited by LEKTI domain 6. Br J Dermatol 2005; 153:12003. 31 Hansson L, Stromqvist M, Backman A et al. Cloning, expression, and characterization of stratum corneum chymotryptic enzyme A skin-specic human serine proteinase. J Biol Chem 1994; 269:194206. 32 Brattsand M, Egelrud T. Purication, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation. J Biol Chem 1999; 274:3003340.

2011 The Authors BJD 2011 British Association of Dermatologists 2011 164, pp13041310