Adapting epicutaneous patch testing protocols to assess immediate-type skin reactions.

OBJECTIVE: During the development of cosmetic formulations, in vitro and in vivo methods are essential tools used to reliably assess the skin irritation potential of a product or ingredient. Epicutaneous patch testing (single and/or multiple application protocols) has long been used as an initial in vivo method to screen for possible skin irritation properties of a substance or formulation. To confirm the mildness and dermatological and/or consumer acceptance of a product, use tests are often subsequently conducted. A study was therefore initiated to see how well patch test results correlate with use tests with respect to irritation elicited by skincare (leave-on) products. METHODS/RESULTS: A number of different cosmetic formulations were assessed in both tests. Although the patch test results did not indicate substantial irritation potentials, immediate-type reactions (stinging and redness) were observed in some volunteers which disappeared within approx. 1 h. Although transient, these reactions suggested that consumer acceptance would probably be low and the studies were discontinued. Immediate-type reactions are rare but have been described for some substances used in cosmetics. These unexpected results were nevertheless intriguing and prompted the start of a journey to see if patch test protocols could be modified to assess these reactions. An occlusive short-term patch test protocol with an application period of 20 min was developed. Successful identification of the spontaneous reactions became possible. Furthermore, there was a correlation between the intensity of reactions observed in the short-term patch test and those observed in the controlled in-use studies. Short-term patch testing using the developed protocol can therefore reliably be used as a screening method, for example in the development and optimization of cosmetic formulations containing ingredients that could cause spontaneous reactions, for instance of non-immunological contact urticaria type. CONCLUSION: The lessons learned from this studies indicate that simple modifications of existing test protocols can lead to important insights into skin reactions. These modifications can then be used to create further building blocks in the development and optimization of test strategies for cosmetic formulations which offer reliable study designs for possible reactions product developers may encounter.

OBJECTIF: Lors du développement de formulations cosmétiques, les méthodes in vitro et in vivo sont des outils essentiels utilisés pour évaluer de manière fiable le potentiel d'irritation cutanée d'un produit ou d'un ingrédient. Le test épicutané (protocoles d'application uniques et / ou multiples) est utilisé depuis longtemps comme méthode initiale in vivo pour dépister les éventuelles propriétés d'irritation cutanée d'une substance ou d'une formulation. Afin de confirmer la douceur et l'acceptation dermatologique et / ou consommateur d'un produit, des tests d'usage sont souvent effectués ultérieurement. Une étude a donc été initiée pour voir dans quelle mesure les résultats des tests épicutanés correspondent aux tests d'usage en ce qui concerne l'irritation provoquée par les produits de soin (sans rinçage). MÉTHODES/RÉSULTATS: Un certain nombre de formulations cosmétiques différentes ont été évaluées dans les deux tests. Bien que les résultats du test épicutané n'indiquent pas de potentiels d'irritation substantiels, des réactions de type immédiat (picotements et rougeurs) ont été observées chez certains volontaires. Celles-ci ont disparu en à peu près 1 heure. Bien que transitoires, ces réactions de type 5 suggéraient que l'acceptation du consommateur serait probablement faible et les études ont été interrompues. Les réactions de type immédiat 6 sont rares mais ont été évoquées en relation avec certaines substances utilisées en cosmétique. Ces résultats inattendus étaient néanmoins intrigants et ont incité le lancement d'un processus pour voir si les protocoles de test épicutané pouvaient être modifiés pour évaluer ces réactions. Un protocole de test épicutané à court terme occlusif avec une période d'application de 20 min a été développé, permettant l'identification réussie des réactions spontanées. Il a été de plus constate une corrélation entre l'intensité des réactions observées dans le test épicutané à court terme et celles observées dans les test d'usage contrôlés. Le test épicutané à court terme utilisant le protocole développé peut donc être utilisé de manière fiable comme méthode de dépistage, par exemple dans le développement et l'optimisation de formulations cosmétiques contenant des ingrédients qui pourraient provoquer des réactions spontanées, par exemple de type urticaire de contact non immunologique. CONCLUSION: Les leçons tirées de ces études indiquent que de simples modifications des protocoles de test existants peuvent révéler des informations importantes sur les réactions cutanées. Ces modifications peuvent ensuite être utilisées pour créer d'autres blocs de construction dans le développement et l'optimisation de stratégies de test pour des formulations cosmétiques qui offrent des conceptions d'études fiables pour les réactions possibles que les développeurs de produits peuvent rencontrer.

Oral intake of specific bioactive collagen peptides reduces skin wrinkles and increases dermal matrix synthesis.

Dietary consumption of food supplements has been found to modulate skin functions and can therefore be useful in the treatment of skin aging. However, there is only a limited number of clinical studies supporting these claims. In this double-blind, placebo-controlled study, the effectiveness of the specific bioactive collagen peptide (BCP) VERISOL® on eye wrinkle formation and stimulation of procollagen I, elastin and fibrillin biosynthesis in the skin was assessed. A hundred and fourteen women aged 45-65 years were randomized to receive 2.5 g of BCP or placebo, once daily for 8 weeks, with 57 subjects being allocated to each treatment group. Skin wrinkles were objectively measured in all subjects, before starting the treatment, after 4 and 8 weeks as well as 4 weeks after the last intake (4-week regression phase). A subgroup was established for suction blister biopsies analyzing procollagen I, elastin and fibrillin at the beginning of the treatment and after 8 weeks of intake. The ingestion of the specific BCP used in this study promoted a statistically significant reduction of eye wrinkle volume (p < 0.05) in comparison to the placebo group after 4 and 8 weeks (20%) of intake. Moreover a positive long-lasting effect was observed 4 weeks after the last BCP administration (p < 0.05). Additionally, after 8 weeks of intake a statistically significantly higher content of procollagen type I (65%) and elastin (18%) in the BCP-treated volunteers compared to the placebo-treated patients was detected. For fibrillin, a 6% increase could be determined after BCP treatment compared to the placebo, but this effect failed to reach the level of statistical significance. In conclusion, our findings demonstrate that the oral intake of specific bioactive collagen peptides (Verisol®) reduced skin wrinkles and had positive effects on dermal matrix synthesis.

Oral supplementation of specific collagen peptides has beneficial effects on human skin physiology: a double-blind, placebo-controlled study.

Various dietary supplements are claimed to have cutaneous anti-aging properties; however, there are a limited number of research studies supporting these claims. The objective of this research was to study the effectiveness of collagen hydrolysate (CH) composed of specific collagen peptides on skin biophysical parameters related to cutaneous aging. In this double-blind, placebo-controlled trial, 69 women aged 35-55 years were randomized to receive 2.5 g or 5.0 g of CH or placebo once daily for 8 weeks, with 23 subjects being allocated to each treatment group. Skin elasticity, skin moisture, transepidermal water loss and skin roughness were objectively measured before the first oral product application (t0) and after 4 (t1) and 8 weeks (t2) of regular intake. Skin elasticity (primary interest) was also assessed at follow-up 4 weeks after the last intake of CH (t3, 4-week regression phase). At the end of the study, skin elasticity in both CH dosage groups showed a statistically significant improvement in comparison to placebo. After 4 weeks of follow-up treatment, a statistically significantly higher skin elasticity level was determined in elderly women. With regard to skin moisture and skin evaporation, a positive influence of CH treatment could be observed in a subgroup analysis, but data failed to reach a level of statistical significance. No side effects were noted throughout the study.

Internet-based lay person rating of facial photographs to assess effects of a cleansing product and a decent cosmetic foundation on the attractiveness of female faces.

It is well established that decorative cosmetics can enhance female facial attractiveness. In this study, we investigated the effects of a cleanser and a decent foundation on attractiveness of female faces. Comparative rating of a set of facial photographs by a group of lay persons revealed that the cleansing product was significantly reducing the attractiveness of the stimulus persons. Treatment with the foundation increased the attractiveness of the female faces clearly. The authors conclude that even unobtrusive cosmetic treatments like cleansers and light foundations may cause relevant changes of the attractiveness of female faces.

Multicentre comparison of skin hydration in terms of physical-, physiological- and product-dependent parameters by the capacitive method (Corneometer CM 825).

A multicentre study for measuring skin hydration with 349 volunteers was carried out in six different laboratories. The purpose of the study was to investigate physical-, physiological- and product-dependent parameters of three test emulsions (base, base + moisturizer and base + moisturizer + lipids) in a double-blind study. A comparison between analogous and digital sensor technology of the Corneometer CM825 was examined. Here, a clear relationship between both sensor types could be highlighted. A vital point of the study was the division of the test subjects according to their skin type. To get more objective limits for three different skin types - very dry, dry and normal skin - visual expert evaluation, self-assessment and hydration measurements were analysed by means of statistical methods. The moisture-related skin types were determined as follows: very dry skin was characterized with corneometer units below 30, dry skin between 30 and 40 and normal skin higher than 40 a.u. (arbitrary units). The efficacy of the three test emulsions was examined in relation to the mentioned skin types. Analysing the measured data of all test centres, a clear dependency of skin physiology (skin type) and product efficacy became evident. The drier the skin, the higher the increase of hydration. The product performance of the three test emulsions compared to the untreated control resulted in a significant increase of skin hydration in all measuring centres. The evaluation of a product ranking showed a good differentiation between the basic emulsion and the two other products. An increase of efficacy by adding lipids could be observed in four of six centres. The important influence of the skin type of the volunteers on the degree of product performance, as demonstrated in this study, should be especially considered when drawing up guidelines for efficacy testing.

Induction of IL-15 messenger RNA and protein in human blood-derived dendritic cells: a role for IL-15 in attraction of T cells.

IL-15 is a pleiotropic cytokine with IL-2-like functions. As IL-15 was shown to be mitogenic for T cells, we wondered whether human blood-derived dendritic cells (DC), as the primary stimulators of T cell responses, are able to produce IL-15. To test our hypothesis, DC were grown under serum-free conditions from human peripheral blood using granulocyte-macrophage CSF and IL-4. Cultures were assayed for IL-15 mRNA production at various times by semiquantitative reverse transcription-PCR. Low baseline signals were detected from days 0 to 5 of culture. A significant increase was detected from days 5 to 9 of the culture. When DC were further enriched by immunomagnetic beads to >98% purity as determined by CD83 staining, IL-15 mRNA signals were exclusively found in the CD83+ fraction. This increase in mRNA signals was paralleled by IL-15 protein release from days 9 to 12 as detected by CTLL-2 assay and ELISA. In addition, protein levels were increased >10-fold by adding paramagnetic beads to the cultures, thereby inducing phagocytic activity. Furthermore, DC supernatants were tested for chemokinetic and chemotactic activities for T cells in a checkerboard filter assay. It was shown that supernatants express chemokinetic and chemotactic activity for T cells. This activity was blocked almost completely by addition of an anti-IL-15 mAb. Our data show that human blood DC contain IL-15 mRNA and produce functional protein that is induced in culture. Protein release is triggered by phagocytic activity. Furthermore, DC-derived IL-15 has chemotactic and chemokinetic activities for T cells, suggesting a role for IL-15 as an attractant of T cells during the initial DC/T cell interaction.

In vitro model for contact sensitization: I. Stimulatory capacities of human blood-derived dendritic cells and their phenotypical alterations in the presence of contact sensitizers.

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in many non-lymphoid tissues and a specialized form of DC-the Langerhans cell (LC)-is found in the skin. The functionality of LC as APC is crucial for the induction of an allergic contact dermatitis. For a long time LC research has been hampered by the limiting numbers of functionally active LC that could be isolated from human skin. The addition of GM-CSF and IL-4 to the non-adherent fraction of mononuclear cells from peripheral blood generated a large amount of CD1a(+) HLA-DR(+) DC. These in vitro-generated DC exhibited the morphology, phenotype and autologous T-lymphocyte stimulating capacity of the human DC/LC system. We had tested phenotypical alterations of in vitro-generated DC under the influence of subtoxic concentrations of different chemicals and contact sensitizers. In vitro stimulation with the contact sensitizers urushiol, primin, C10-and C11-primin analogues, alantolactone, isoalantolactone and NiSO(4) resulted in a decrease of HLA-DR expression on the surface of these cells if the incubation period did not exceed 3 hr. Incubation with irritants like sodium lauryl sulfate (SLS) and benzalkonium chloride did not change or increase the HLA-DR surface expression under these conditions. With regard to the adhesion molecule ICAM-1, there was no clear difference between irritants and contact sensitizers. But based on the alteration of HLA-DR expression of dendritic cells under short-term exposure conditions, there was a clear-cut difference between irritants and contact sensitizers. In summary, this system can be used to discriminate between contact sensitizers and irritants.

In vitro model for contact sensitization: II. Induction of IL-1beta mRNA in human blood-derived dendritic cells by contact sensitizers.

Epidermal mRNA for interleukin 1beta (IL-1beta) has been shown to be increased following exposure of mouse skin to sensitizing compounds. In addition, this early upregulation of IL-1beta was specific for contact sensitizers, while expression of IL-1beta was unaffected by irritants. Langerhans cells are the major source of IL-1beta within the epidermis in the induction phase of skin sensitization. Since the isolation of Langerhans cells from skin biopsies results only in low frequencies, we decided to use dendritic cells (DCs) generated from peripheral blood as Langerhans cell equivalents to investigate the ability of five contact sensitizers and one irritant to induce IL-1beta gene expression in vitro. For our studies we cultivated DCs in serum-free medium supplemented with granulocyte/macrophage-colony stimulation factor (GM-CSF) and interleukin 4 (IL-4). The DCs showed a typical dendritic morphology, a characteristic expression of surface markers and high stimulatory capacity for autologous T cells. 5-day-old DCs were incubated with subtoxic concentrations of the contact sensitizers pentadecyl-catechol, 2,4,6-trinitrobenezene sulfonic acid, 2,4-dinitrofluorobenzene, NiSO(4), K(2)Cr(2)O(7) and the irritant sodium dodecyl sulfate. IL-1beta mRNA expression was detected by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and non-radioactive hybridization procedures. For all contact sensitizers, expression of IL-1beta mRNA increased, whereas treatment with the irritant SDS had no significant effect on IL-1beta expression. Thus we developed an in vitro system, which may be useful to evaluate allergic potentials of chemicals and products.

In vitro analysis of immunoprotective effects of topical sunscreens.

The aim of this study was to evaluate procedures for the assessment of the immunoprotective capacities of topical sunscreens, using cell cultures. The exposure of humans to solar or ultraviolet radiation has been shown to induce numerous changes at the level of the immunoresponsiveness of the skin, which could be described as immune suppression. In this study, a sunscreen containing the commercially available UVB filters octyl triazone, phenylbenzimidazole sulfonic acid and methylbenzylidene camphor, was tested because of its capacity to protect from the immunosuppressive effects of UVB radiation. In contrast to control suncreens with a comparable sun protector factor (SPF), but that did not contain the UVB-filter octyl triazone, the immunoprotective sunscreen protected from two forms of immune suppression as assessed in vitro. Expression of the immune-relevant cellular communication structure intercellular adhesion molecule-1 is decreased on the cell surface of epidermal cells under the influence of UVB. This effect of immune suppression is totally abolished by the immunoprotective capacity of the tested sunscreen, as monitored cytofluorometrically. The mixed-lymphocyte reaction (MLR) was also used to assess the protective capacities of sunscreens against UVB-radiation-induced suppression of the immune response. Again, this UVB-induced suppression of the MLR was prevented by the sunscreen containing octyl triazone. In summary, the results of this study demonstrate the usefulness of the above-mentioned methods for the in vitro evaluation of the immunoprotective properties of topical sunscreens.

Immunocompetent cells for in vitro screening of skin irritation.

The present studies were aimed at evaluating procedures for assessing the immunmodulatory effects of chemicals and preparations on macrophage differentiation and lymphocyte proliferation in cell cultures. The effects of 10 drugs and anti-inflammatory agents were monitored by determining thymidine incorporation into phytohaemagglutinin (PHA)-stimulated T cells in the lymphocyte transformation test (LTT) and the expression of two surface antigens on macrophages in the macrophage differentiation assay (MDA). One antigen was found on macrophages in acute inflamed tissue. The other was detected on those found in recovering tissue. These parameters were compared with mean skin irritation scores for 12 known cosmetic products from epicutaneous patch testing. Finally, these parameters were also used to study six cosmetic test formulae with unknown irritation potentials subjected to blind testing during phase 2 of the "CTFA Evaluation of Alternatives Program". Immunosuppressive agents were detected in both systems. Agents, thought to be pro-inflammatory, were monitored in the MDA by the acute inflammation marker. Skin irritation scores of known preparations correlated well with those of expressed acute inflammation markers in the MDA (r(s) = 0.714), but no clear relationship was detectable in the LTT. In contrast one of the CTFA samples tested blind revealed a strong response in both tests. The roll-on antiperspirant stimulated T-cell proliferation and induce a strong expression of the acute inflammation marker on macrophages. Based on these findings further studies are in progress to evaluate the usefulness of these in vitro tests for predicting dermal irritation.

T cell receptor alpha and beta gene expression in a murine antigen-specific T suppressor lymphocyte clone with cytolytic potential.

The composition of alpha and beta TCR genes was analyzed in a murine BSA-specific Ts cell clone with cytolytic potential. The isolated poly(A)+ mRNA from Ts cell clone BVI/5 was used to construct a cDNA library in the bacteriophage lambda gt11. Full-length cDNA clones specific for TCR alpha and TCR beta genes have been detected and isolated by hybridization with specific oligonucleotide probes. The functional rearranged TCR alpha gene is composed of a member of the V alpha 1 family, the junctional gene J alpha TT11 and C alpha. The gene segments V beta 13, D beta 2.1, J beta 2.4, and C beta 2 form the functional rearranged TCR beta gene. Furthermore, a nonfunctional TCR alpha gene transcript has been detected, where a V alpha 8 gene is rearranged to a so far not described J alpha gene segment (J alpha BVI). A stop codon in its junctional region is responsible for this non-functional transcript. By using Southern blot analysis, the described rearranged TCR genes can be detected in the J alpha junctional region and in the J beta 2 cluster on the genomic DNA level. Immunoprecipitation studies with the KT3 anti-CD3 mAb and flow microfluorimetry analysis with the H57-597 anti-TCR-alpha/beta mAb show that TCR/CD3 complexes are synthesized and expressed on BVI/5 Ts cells. Taken together, the cDNA sequencing data, the protein studies, and the specificity of Ag recognition demonstrate that the BVI/5 Ts cell clone not only transcribes the TCR alpha and beta genes but also expresses a functional BSA-specific receptor.

Specific in vivo suppression of lymph node cell proliferation and humoral immune responses by cloned antigen-specific T suppressor cells.

Two BSA-specific Ts cell clones have been isolated from CBA/J mice tolerized by low doses of BSA. Together with one Ts cell clone isolated from an immune animal, they have recently been characterized with regard to phenotypes and in vitro functions. In the present report the in vivo effector functions of two of them (Ts cell clones BVI/5 and HF1.MS) are described. BSA-primed lymph node cells from CBA/J mice, which had received BVI/5 or HF1.MS Ts cells at the time of immunization, do not respond to a subsequent in vitro antigenic challenge. A human gamma-globulin (HGG)-specific lymph node cell proliferation is not influenced. BVI/5 Ts cells injected into mice at the time of priming with fluorescein (Flu)-conjugated BSA or Flu-HGG inhibit the humoral anti-Flu-response in Flu-BSA-primed animals. The anti-Flu-response in Flu-HGG-primed animals is only marginally affected by BVI/5 Ts cells. The data show that it is possible to induce immunologic unresponsiveness at the humoral level by reexposing in vitro propagated Ts cell clones to their syngenic in vivo environment.

An evaluation of the existence of soluble suppressor factors in cloned antigen-specific T suppressor lymphocytes.

The suppressive activity of two bovine serum albumin-specific class II-restricted T suppressor cell clones (BVI/5 and 83/2-D11) was compared to that of a feeder cell-independent, IL 2-dependent subline (HF1.IL-2) of an originally antigen-dependent class II-restricted Ts cell clone (HF1). No soluble suppressor factors can be found in BVI/5 and 83/2-D11 Ts cell extracts or culture supernatants under conditions where an unspecific factor can be derived from HF1.IL-2 cells. This factor, when isolated from cell extracts in the presence of n-octyl-beta-D-glycopyranoside, has a molecular weight of 70-80 kD. In absence of this non-ionic detergent, it has a high affinity to membrane fragments and is associated with a molecular weight of 300 kD or more. The data are discussed in connection with recent findings of direct T suppressor to T helper interaction by cell-cell contact.

Isolation of a bovine serum albumin-specific T-suppressor cell clone and evaluation of its in vitro functions.

This communication describes a newly isolated T-suppressor (Ts) cell clone, BVI/5, derived from a CBA/J mouse tolerized to low doses of bovine serum albumin (BSA). The cells are Thy-1.2+, Lyt-1.1-, Lyt-2.1+, and express endogenous derived I-Ak and I-Ek molecules. BVI/5 Ts cells show antigen-specific I-Ek-restricted proliferation in the presence of antigen-presenting spleen cells. This proliferation can be inhibited by anti-I-Ek but not by anti-I-Ak monoclonal antibodies. In vitro antigen-specific suppressive capacity has been measured in a primed lymph node cell proliferation assay, and the fine specificity of these suppressor functions has been studied. Broader cross-reactivity in the specificity of this effector function is found if compared to the specificity of antigen-induced proliferation. The data extend previous findings on the expression of I-A and I-E molecules on Ts cells and are discussed with respect to the role of those cells in tolerance induction.

Expression of L3T4 molecules on Lyt-2+, class II-restricted antigen-specific T suppressor lymphocytes.

Three bovine serum albumin-specific Lyt-2+ T suppressor (Ts) cell clones from CBA/J mice have been analyzed with regard to expression of L3T4 molecules. All three Ts-cell clones can be stained with monoclonal antibodies (mAb) to L3T4. Tested for the two clones restricted to recognition of Ek determinants, antigen-specific proliferation on antigen-presenting cells, but not the proliferation induced by conditioned medium can be inhibited by L3T4-specific mAb. In a similar way, Ts-cell cytolytic effector functions can be blocked by L3T4-specific mAb. Thus L3T4 structures seem to play a role in Ts-cell functions. Furthermore, the data support the view that L3T4 expression can be a property of class II-restricted T cells irrespective of their Lyt phenotype.