Increase of skin-ceramide levels in aged subjects following a short-term topical

application of bacterial sphingomyelinase from Streptococcus thermophilus.

Maurizio Giuliani*, Benedetta Cinque§, Luisa Di Marzio°, Gianfranco Amicosante^ e

M. Grazia Cifone§

§Dept. Experimental Medicine, University of L’Aquila.

°Dept. Drug's Science, University G. D'Annunzio, Chieti Scalo, Italy.
*Dept. Surgical Sciences, University of L’Aquila.
^Dept. Biomedical Sciences and Technologies

Abstract: Several studies have demonstrated that ceramides play an essential role in both
the barrier and water-holding functions of healthy stratum corneum, suggesting that the
dysfunction of the stratum corneum associated with ageing as well that observed in patients
with several skin diseases (i.e. atopic dermatitis, psoriasis) could result from a ceramide
deficiency. In a previous study, our group have reported a significant increase in skin
ceramide levels in healthy subjects, after a treatment in vivo with a cream containing a
preparation of S. salivarum subspeciem thermophilus, a probiotic belonging to the lactic acid
bacterium (LAB) group. The presence of high levels of neutral sphingomyelinase activity
(nSMase) in this organism was responsible for the observed increase of stratum corneum
ceramide levels, thus leading to an improvement in barrier function and maintenance of
stratum corneum flexibility. Recently, we have also reported the beneficial effects on atopic
dermatitis patients of a topical treatment with a cream containing S. thermophilus. The
experimental cream, also in this case, was shown to induce a relevant increase of skin
ceramide levels which was associated to an improvement in the clinical signs and symptoms
of atopic dermatitis. The aim of the present work was to investigate the effects of the topical
treatment of an S. thermophilus-containing cream on ceramide levels of stratum corneum
from healthy aged patients. A 2-week application of the cream, containing a sonicated
preparation of the lactic acid bacterium S. thermophilus, in the forearm skin of 20 patients led
to a significant and relevant increase of skin ceramide amounts, which could have resulted
from the sphingomyelin hydrolysis through the bacterial sphingpomyelinase. The topical
application of a sonicated Streptococcus thermophilus preparation, leading to increased
stratum corneum ceramide levels, could thus result in the improvement of lipid barrier and a
more effective resistance against ageing-associated xerosis.

Introduction
The stratum corneum (SC), a highly specialized structure, is the outermost layer of the skin
and functions as an important barrier to maintain biological homeostasis. The SC is
essentially impermeable to water except for a small but vital flux that serves to maintain its
hydration, and thereby, its flexibility. Hydration of the surface layers is also critical to facilitate
desquamation, the process of skin shedding at the skin surface [1]. The unique structure of
the stratum corneum is well established. It is comprised of both nonviable, protein-enriched
corneocytes and a surrounding lipid-enriched extracellular matrix [2]. The intercellular lipids
are formed, in part, by small, ovoid vesicles located in the cells of the stratum granulosum
layer: the lamellar bodies [3]. The lamellar bodies are extrused by exocytosis through the cell
membranes. Their contents then migrate to the outer epidermis where they interdigitate
between the corneocytes to form a watertight barrier at the level of the stratum granulosum.
The lamellar bodies are composed predominately of lipids, including cholesterol, ceramides
and fatty acids [4] tipically with ratios in the range of 3:4:2, respectively [5]. All three
components are required for skin integrity [6-8]. However, it is the ceramides that are thought
to play the essential role in the formation of the bilayer system. The extracellular Cer
comprise ~ 50% of the SC lipids [9, 10], and represent a heterogeneous family of at least
seven molecules, with variation in the long-chain sphingoid base structure, as well as in the
chain length and alfa-hydroxylation of constituent amide-linked FA.
A multitude of factors, including disease, age, diet, race, and of course, the external
environment, may render the barrier more prone to perturbation and potentially induce
dryness, irritation, or itch [11]. In the case of Caucasians it has been shown that levels of
ceramides diminish with increasing age, whereas the ratio between the ceramides subtypes
remains constant [12]. The amount of ceramide in the stratum corneum is regulated by the
balance among the ceramides generating enzymes including serine-palmitoyltransferase [13],
sphingomyelinase (SMase) [14, 15], and b-glucocerebrosidase [16], and the degradative
enzyme ceramidase [17]. Both sphingomyelin and sphingomyelinase, are better able to
hydrolyse sphingomyelin into ceramides, are present in the epidermis and are originally
contained in lamellar bodies [18]. In our previous work we assessed the possibility of
increasing ceramide levels either in vitro, on cultured keratinocytes, or in vivo, on stratum
corneum by treatment with a cream containing a sonicated preparation of Streptococcus
salivarius subspeciem thermophilus [19]. The results showed that S. thermophilus treatment
led to a relevant increase of ceramide levels both in vitro and in vivo in healthy subjects. Our
group, recently, has assessed the beneficial effects of topical administration of a S.
thermophilus-containing cream, for two weeks, on ceramide levels of SC from atopic
dermatitis patients [20]. Considering the suggested role of the ceramides in regulating the
water-holding capacity and in maintaining skin integrity, the aim of the present work was to
investigate the possibility that the topic application of S. thermophilus, representing a source
of exogenous SMase able to hydrolyze skin SM and consequently to generate ceramides,
presumably may lead to reduce dryness, loss of tone, fullness and water loss.

Materials and methods

Preparation of experimental cream. Streptococcus thermophilus (Strain S244) cultivated in
10% skimmed-milk sterilized at 110°C for 30 min and added to 0.1% yeast extract was
obtained from VSL Pharmaceuticals (Gaithersburg, Maryland) in a pure lyophilized form (108
colony-forming units [CFU]/g). Stocks of 1.7-g lyophilized S. thermophilus were resuspended
in 5 ml of phosphate buffered solution (PBS), sonicated (30 min, alternating 10 s sonication
and 10 s pause) with a Vibracell sonicator (Sonic and Materials Inc., Danbury, CT). For the
topical applications, the sonicated bacteria (1.7 g/5 ml) were firstly analyzed for the neutral
SMase activity and then mixed with 20 ml of a base cream (Avant Garde, Sigma Tau,
Pomezia, Rome, Italy) containing the following components: Sepigel 305, Vitamin E acetate,
perfume, glycerol, vaselin oil, Glucam E 20, Fenotan, EDTA, Paracombin, Carbopol ETD
2001, Bensil dm 350, imidazolidynil urea, trietanolamine, Transcutol, demineralised water.

Study design. The study was conducted on 20 healthy Caucasian females (68 ± 3 years)
with healthy skin. At the beginning of the study each volunteer signed the informed consent
draw up by the technicians. The eligibility criteria used to include subjects in this study were
the following: Caucasian race; major 60 year’s old, healthy skin, mentally competent to
complete the treatment. Exclusion criteria were: history of intolerance to drugs and/or
cosmetic products and mild-moderate atopic or contact dermatitis. For the whole duration of
the study the subjects must not use different products on the tested areas and must avoid the
exposure to UV radiation. The trial was conducted during the autumnal season in order to
avoid interseasonal variations of the skin ceramide content. During the period of the trial no
other drug, topical or systemic, was allowed. Each subject applied twice daily for 15
consecutive days an ‘active’ formulation (base cream as vehicle containing S. thermophilus)
to one forearm (treated site, T) and the contralateral forearm was left untreated and served as
control (untreated site, UT).
At the beginning of the study (time T0) and at the end of application period (time Tf) the
biochemical and instrumental evaluations were performed on each investigated site. The
study was carried out in a bioclimatic room (24°C; 50% rh) in order to keep constant the
temperature and the humidity during the measurements. Each volunteer was required not to
either cleance or moisturice the forearms for 4 hours prior to the beginning of the test.

Corneometer measurements
Skin hydration was investigated using a Corneometer CM 825 (Courage und Khazaka,
Germany) which was mounted on a Multi Probe Adapter MPA 5 (Courage und Khazaka,
Germany). Capacitance changes depending almost solely upon the water content in the
stratum corneum are detected and evaluated. The conductivity of this closed system changes
as a function of stratum corneum hydration. Three readings were taken in contiguous points
of each testing area on the volar forearm. The results are given in “arbitrary units” (arb. units).
[21]

Tewameter measurements
Barrier function was evaluated by measuring the transepidermal water loss (TEWL) (g/cm2h)
using the Tewameter TM 210 (Courage und Khazaka, Germany). In accordance with
applicable guidelines [22]. TEWL is considered an important measure of epidermal barrier
function. Evaporimetry consists of applying a probe with two twin sensors directly to the skin,
with one sensor pair measuring humidity and the other temperature. The acquired data are
used by an integrated microcomputer to compute the water vapour partial pressures at the
two parallel levels of each sensor pair and, via the partial pressure gradient, the rate of
evaporation. To minime outside interference, the measurements were carried out in an open-
top Plexiglas chamber with closed sides .

Sample collection. To assess the skin ceramide levels, stratum corneum sheets were
removed from the volar aspect of the forearms (uncleaned before sampling), 10 cm below the
antecubital fossa, by a single stripping with 25 cm2 of cyanoacrylate resin, before (T0) and
after 2 weeks of the application (Tf). The cyanoacrylate resin was immersed for 1 h in 25 ml of
chloroform: methanol (2:1, by vol.), after which cyanoacrylate resin was removed. The
samples were filtrated with a 0.45 µm micropore filter (Millipore Corporation Bedford, MA
USA) and concentrated in a rotary evaporator (Savant Instruments Inc., Holbrook, NY) and
evaporated to dryness in glass tubes under a stream of nitrogen, and the residues were
dissolved in 1 ml of chloroform/ methanol (9/1, by vol) and stored at 20°C until use.

Analysis of stratum corneum ceramides. For the lipid extraction, 400 µl of methanol, 500 µl
of chloroform and 400 µl of water were added. Samples were stirred for 2 min on a vortex-
mixer and centrifuged at 10978xg for 10 min. The extraction and centrifugation steps were
repeated twice. Lipids, previously dried under nitrogen, were then incubated with Escherichia
coli diacylglycerol kinase (DAG kinase assay kit and 32P-ATPgamma, spec. act. 3Ci/mmol,
Amersham, Buckinghamshire, UK) to determine the levels of ceramide, according to the
manufacturer’s instructions, and applied to thin layer chromatography (TLC) silica gel plates
using a TLC applicator (Camag; Berlin, Germany). Ceramide phosphate was then resolved
using CHCl3/CH3OH/CH3COOH (65/15/5, v/v/vol) as solvent. Authentic ceramides from
bovine brain (Ceramide Type III, non-hydroxy fatty acid ceramides; and Ceramide type IV,
hydroxy fatty acid ceramides, Sigma) were identified by autoradiography at Rf=0.25 and
Rf=0.11, respectively. Specific radioactivity of ceramides-1-phosphate was determined by
scintillation counting of corresponding spots scraped off the gel. Quantitative results for
ceramide production were obtained by comparing the experimental values with linear curve of
the ceramide standards, and are expressed as pmoles of ceramides-1-phosphate/cm2.

Statistical analysis
Mean value and standard deviation were calculated for each set of instrumental and
biochemical values (basal and final) and for each parameter relating to the tested skin areas.
Furthermore, it was calculated the variation of the parameter (Tf-T0; Tf = mean value at the
end of the treatment and T0 = mean value at the beginning of the treatment). The basal and
the final instrumental values of the investigated areas were statistically compared by t-test.
The variations (Tf vs T0) occurred in the treated and untreated sites of the forearms were
statistically compared by Wilcoxon test. The group of data were considered significantly
different for a probability value p≤0.05. The Student’s t-test for paired data was used to
analyse the differences between the ceramide levels in the subjects before (T0) and after (Tf)
treatment with the experimental cream. The STATPAC Computerized Program was used to
perform statistical analysis, and a P value ≤0.05 was used as the significance criterion.

Results
Effect of experimental cream on skin hydration and barrier function. To evaluate the
efficacy of the experimental cream on healthy human aged skin, biophysical measurements
were performed, on a group of aged subjects, before (T0) and after two weeks (Tf) of the
treatment. The barrier function was determined by evaluating the transepidermal water loss
(TEWL) (figure 1). No statistically significant change of TEWL values was found on treated
(T0: mean±SEM=10±2.6; Tf: mean± SEM=10.4±4.7) and untreated sites of the forearms (T0:
mean± SE.M=10±3.3; Tf: mean± SEM.=10±2.6). The statistical comparison of the variations
obtained in the treated and the untreated sites did not show any significant difference (T site:
Variation % = +4%; UT site: Variation % = 0, p> 0.05). This means that variations in
transepidermal water loss are too small to be taken into consideration. On the other hand,
significant difference was obtained in skin capacitance (figure 2). During the application
period, the active-formulation increased the mean skin hydration from 37.0±7.3 (mean± SEM)
to 41.2±6.7 (mean±SEM), whereas the untreated sites remained unchanged (T0: mean±
SEM=36.3 ±6.7; Tf: mean± SEM=35.7±6.2). A statistically significant increase of hydration
values was found on the treated site (T site: Variation % = +11.3%; p <0.05), while no
significant change was observed on the control untreated site (UT site: Variation % = -1.6%).
The statistical comparison of the variations obtained in the treated and the untreated sites
showed a highly significant difference among them (p=0.001).

Ceramide levels in the healthy aged subjects. In order to determine whether the treatment
with S. thermophilus-containing cream was able to affect skin ceramide levels of healthy aged
subjects, we analyzed the amounts of ceramide from lipid extracts from the forearms of 20
aged volunteers, before (T0) and after two weeks (Tf) of the treatment. In table I are showed
the ceramide levels at the beginning (T0) and at the end (Tf) of the treatment for two weeks
and the percent increase values. The skin ceramide levels of healthy aged subjects showed
strong basal subjective variability (range:0.2-40 pmol total ceramides/cm2;
mean±SEM.=9.07±2.92 pmol total ceramides/cm2). Total stratum corneum levels were
significantly increased in almost all subjects following topical application of “active”
formulation (range:2-460.60 pmol total ceramides/cm2; mean±SEM.=61.65±25.9 pmol total
ceramides/cm2; Tf versus T0, p=0.053).

Discussion
The skin barrier and the water-holding capacity are the most important functions of the SC
and these functions are related to the composition and structure of SC intercellular lipids [23,
24], including cholesterol, ceramides and fatty acid. The role of ceramides in the horny layer
of human skin has been investigated by numerous studies [25, 26]. After initial studies
detailing the lipid composition of the epidermis and its upper layers [27], much work was done
on possible correlations between ceramide content and distribution and moisture retention,
formation of scaly skin, age, pathological states and reaction of the skin to external factors
[23, 28].
The presented study was aimed to study whether S. thermophilus extracts, which showed to
express high levels of neutral SMase activity [19] could exert a beneficial effect on the
properties and characteristics of the aged skin by measuring its effect on the TEWL,
capacitance and ceramide levels. Our results showed that the topical application of S.
thermophilus-containing cream improved properties of dry and aged healthy human skin,
whereas a non-significant modification of transepidermal water loss was observed. This
evidence confirmed previously reported findings, which showed that no change in TEWL has
been shown following treatment with moisturizers in vivo although increased hydration was
found [29, 30]. The cause of no significant change in TEWL in healthy treated and untreated
sites are unknown. Possible causes of unchanged TEWL could include decreased sweating,
decreased skin temperature or increased corneocyte cell surface area. Although neither of
these factors alone can account for the no variation of TEWL in aged epidermis, a
combination of these factors could be operative.
In the light of our findings, the aged subjects seems to be sensitive to barrier improvement
(resulting in a prompt increase in the water-holding capacity) when S. thermophilus-containing
cream was applied. In fact at the end of treatment a statistically significant increase in
hydration values was showed when compared with the values observed at the beginning. As
expected, the increase of the stratum corneum ceramides levels resulted in an improvement
in hydration skin. These results could be explained with the presence of high levels of neutral
SMase in S. thermophilus, which could hydrolyse skin sphingomyelinase thus generating
ceramides. The topical application of a sonicated Streptococcus thermophilus preparation,
leading to increased stratum corneum ceramide levels, could thus result in the improvement
of lipid barrier and a more effective resistance against ageing-associated skin xerosis.
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Figure 1. TEWL values in untreated (UT) and S. thermophilus extract-containing cream
treated (T) subjects (mean± SEM. n. 20, p>0.05). The activity of the product on
transepidermal water loss on the investigated sites is showed. The TEWL values were
determined at the beginning (T0) and at the end (Tf) of a two week treatment.
Figure 2. Alteration in skin capacitance values in untreated (UT) and S. thermophilus extract-
containing cream treated (T) subjects (mean± SEM, n. 20 *p=0.001). Capacitance was
measured in the investigated areas at the beginning (T0) and at the end (Tf) of a two week
treatment.
Figure 3. Effects of S. thermophilus extract-containing cream on stratum corneum ceramide
levels in 20 healthy aged subjects. A) Forearm skin lipid extracts before (T0) and after (Tf) a
two week treatment were analyzed for ceramide level determination by diacylglycerol (DAG)
kinase assay. B) Percent increase of skin ceramide levels after the experimental treatment.