Emollient structure and chemical functionality effects on the biomechanical function of human stratum corneum.

OBJECTIVE: Cosmetic emollients are widely used in skincare formulations due to their ability to 'soften' the skin and modulate formulation spreadability. Though emollients are commonly used, little is known about their effects on the biomechanical barrier properties of human stratum corneum (SC), which play a critical role in consumer perception of formulation efficacy. Accordingly, our objective was to provide new insights with a study involving fourteen cosmetic emollient molecules with widely varying structures, molecular weights, SC diffusivities, topological polar surface areas (TPSAs), viscosities and chemical functionalities. METHODS: Mechanical stress in the SC was measured in vitro using a substrate curvature measurement technique. Stress development due to SC drying was measured before and after topical treatment with cosmetic emollients. Emollient diffusivity and alterations to lipid content in SC after treatment were measured via ATR-FTIR spectroscopy. The maximum penetration volume of emollient in SC was characterized to elucidate mechanisms underlying emollient effects on stress. RESULTS: The application of all cosmetic emollients caused a reduction in SC mechanical stress under dehydrating conditions, and a linear correlation was discovered between emollient penetration volume and the degree of stress reduction. These molecules also induced increases in stress equilibration rate, signalling changes to SC transport kinetics. Stress equilibration rate increases linearly correlated with decreasing intensity of the νCH2 band, indicating a previously unknown interaction between cosmetic emollients and SC lipids. Stress and penetration volume results were rationalized in terms of a multi-parameter model including emollient molecular weight, diffusivity, TPSA and viscosity. CONCLUSION: We provide a new rational basis for understanding the effects of cosmetic emollient choice on biomechanical properties affecting SC barrier function and consumer perception. We demonstrate for the first time that emollients very likely reduce SC mechanical stress through their ability to take up volume when penetrating the SC, and how molecular weight, SC diffusivity, TPSA and viscosity are predictive of this ability. As cosmetic formulations continue to evolve to meet the needs of customers, emollient molecules can be selected that not only contribute to formulation texture and/or spreadability but that also leverage this novel connection between emollient penetration and SC biomechanics.

OBJECTIF: Les émollients cosmétiques sont largement utilisés dans les formulations de soins de la peau en raison de leur capacité à «adoucir¼ la peau et à moduler la capacité d'étalement de la formulation. Bien que les émollients soient couramment utilisés, on en sait peu sur leurs effets sur les propriétés de barrière biomécanique de la couche cornée humaine (SC), qui jouent un rôle essentiel dans la perception par les consommateurs de l'efficacité de la formulation. En conséquence, notre objectif était de fournir de nouvelles perspectives avec une étude impliquant quatorze molécules émollientes cosmétiques avec des structures, des poids moléculaires, des diffusivités SC, des surfaces polaires topologiques (TPSA), des viscosités et des fonctionnalités chimiques très variables. MÉTHODES: La contrainte mécanique dans le SC a été mesurée in vitro en utilisant une technique de mesure de la courbure du substrat. Le développement du stress dû au séchage SC a été mesuré avant et après un traitement topique avec des émollients cosmétiques. La diffusivité émolliente et les altérations de la teneur en lipides dans la SC après le traitement ont été mesurées par spectroscopie ATR-FTIR. Le volume de pénétration maximal de l'émollient dans SC a été caractérisé pour élucider les mécanismes sous-jacents aux effets émollients sur le stress. RÉSULTATS: L'application de tous les émollients cosmétiques a entraîné une réduction de la contrainte mécanique SC dans des conditions de déshydratation, et une corrélation linéaire a été découverte entre le volume de pénétration de l'émollient et le degré de réduction de la contrainte. Ces molécules ont également induit des augmentations du taux d'équilibrage des contraintes, signalant des changements dans la cinétique de transport SC. Le taux d'équilibrage des contraintes augmente linéairement en corrélation avec la diminution de l'intensité de la bande νCH2 , indiquant une interaction jusque-là inconnue entre les émollients cosmétiques et les lipides SC. Les résultats du stress et du volume de pénétration ont été rationalisés en termes d'un modèle multi-paramètres comprenant le poids moléculaire émollient, la diffusivité, le TPSA et la viscosité. CONCLUSION: Nous fournissons une nouvelle base rationnelle pour comprendre les effets du choix des émollients cosmétiques sur les propriétés biomécaniques affectant la fonction de barrière SC et la perception du consommateur. Nous démontrons pour la première fois que les émollients réduisent très probablement la contrainte mécanique SC grâce à leur capacité à prendre du volume lors de la pénétration du SC, et comment le poids moléculaire, la diffusivité SC, le TPSA et la viscosité sont prédictifs de cette capacité. Alors que les formulations cosmétiques continuent d'évoluer pour répondre aux besoins des clients, des molécules émollientes peuvent être sélectionnées qui contribuent non seulement à la texture et / ou à l'étalement de la formulation, mais qui exploitent également cette nouvelle connexion entre la pénétration des émollients et la biomécanique SC.

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.

In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

Peptide reactivity associated with skin sensitization: The QSAR Toolbox and TIMES compared to the DPRA.

The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies. Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in 'no alert' for 83%-100% of the non-sensitizers, but alerted only 55%-61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new "DPRA profilers" (QSAR Toolbox) provided unsatisfactory results. Additionally, the results were combined in the '2 out of 3' prediction model with in vitro data derived from LuSens and h-CLAT. Using DPRA results, the model identified 90% of human sensitizers and non-sensitizers; using in silico results (including abiotic and biotic activations) instead of DPRA results led to a comparable high predictivity.

Restoration of joint congruency and the glenoidal labrum after arthroscopic revision Bankart repair: a MRI match-paired analysis comparing primary Bankart repair and the uninjured labrum.

PURPOSE: The restoration of joint congruency and labrum slope and height after arthroscopic revision Bankart repair (RB) compared to the primary arthroscopic Bankart repair (PB) remain unclear. METHODS: Twenty-three consecutive patients after RB with minor glenoid deficits were matched to 23 patients after PB and retrospectively followed by a score system and native 1.5 T magnetic resonance imaging (MRI) assessment. Bankart repair surgeries were performed using double-loaded knotless suture anchors. The glenoidal (GAA) and labral articulation arc (LAA), labrum slope, height index and morphology were assessed separately for the anterior and inferior glenoid and compared to 23 healthy volunteers [radiologic control group (RC)]. RESULTS: Arthroscopic revision Bankart repair showed 28.0 months post-operative equivalent anterior labral congruency (LAA, 9.3°/PB 9.9°/RC 10.1°) and inferior (LAA 9.9°/PB 9.6°/RC 10.5°). The anterior GAA remain decreased (54.6°/PB 55.7°/RC 58.0°) with an original inferior GAA (85.1°/PB 83.2°/RC 83.8°). The RB labrum was slightly decreased anteriorly (slope 22.9°/PB 23.9°/RC 24.6°; height index 2.4/PB 3.0/RC 3.2). The inferior portion had an equivalent labrum slope (23.8°/PB 24.7°/RC 25.1°), but a decreased height index (2.1/PB 2.2/RC 2.3). Morphologic labrum analysis revealed significant changes between all three groups. The clinical outcome after revision surgery was good-to-excellent, but inferior to the primary stabilization and without influence of joint congruency and labrum morphology to the clinical outcome. CONCLUSION: A properly applied arthroscopic revision of a Bankart repair generates sufficient restoration of the anteroinferior labral joint congruency and good clinical results. STUDY DESIGN: Case series.

[Therapy of degenerative and posttraumatic rotator cuff lesions]. / Therapie von Degenerationen und Verletzungen der Rotatorenmanschette.

The prevalence of rotator cuff lesions is age-dependent and up to 19-32 % for full-thickness ruptures and 13-32 % for partial-thickness lesions respectively. The therapy of partial-thickness ruptures should be considered in accordance with the articular, bursal or intratendinous location of the lesion. The therapy of full-thickness ruptures should be applied in accordance with topography and area of defect, retraction, atrophy and fatty infiltration. These parameters are considered to be important prognostic factors for the intraoperative repairability and the success of the surgery. Symptomatic or chronically progredient partial-thickness lesions as well as full-thickness lesions should generally be treated by means of surgical reconstruction. No current scientific consensus exists regarding improved clinical outcome data after the surgical approach in mini-open or arthroscopic technique. Both procedures should meet the requirements of the Gerber criteria for rotator cuff reconstruction: high primary stability, reduction of micro-movements, minimized approach associated morbidity and persisting stability to enable the fibroblastic tendon-to-bone healing. Current studies revealed a potential improvement of the tendon-to-bone healing by the application of several biologic augmentations. At the moment, these additive procedures can be applied in revision situations and for complex rotator cuff lesions with low tendon quality. No high-level in-vivo investigations concerning the human shoulder exist in the current literature that show evidence-based improvements by the additively applied biologic augmentations for rotator cuff repair.

[Treatment of phalangeal fractures using the mini-hook plate. An alternative for surgical fixation of small phalangeal bone fragments?]. / Versorgung von Fingerfrakturen mit der Minihakenplatte. Alternative in der operativen Versorgung von knöchernen Ausrissen an den Phalangen?

BACKGROUND: Fixation of the small bony fragments of the phalanges is often difficult. In this study a clinical and radiological evaluation was carried out after operative treatment using the mini-hook plate. PATIENTS AND METHODS: Between 2003 and 2006 a total of 36 fractures were treated operatively using the mini-hook plate. Of the patients 24 had an basal avulsion fracture of the distal phalanx and 11 patients (12 fractures) had other bony avulsion fractures of the phalanges. The patients were evaluated clinically and radiologically as well as using the disabilities of the arm, shoulder and hand (DASH) questionnaire. RESULTS: A total of 29 patients with 30 fractures were examined. The mean follow-up was 13.6 months. The mean range of motion in the affected finger joint was 60.3 ° and the mean DASH score was 2.8 points. Postoperatively five nail growth defects, one infection and one secondary dislocation of the implant were observed. CONCLUSION: Using the mini-hook plate, preservation of the joint and stable internal fixation with no need for temporary arthrodesis is possible; however, prerequisites are experience and skill of the surgeon with a difficult surgical technique.

Intraoperative C-arm CT imaging in angular stable plate osteosynthesis of distal radius fractures.

The purpose of this study was to analyze the practicability and benefit of intraoperative C-arm computed tomography (CT) imaging in volar plate osteosynthesis of unstable distal radius fractures. During a 1 year period, intraoperative three dimensional (3D) imaging with the ARCADIS Orbic 3D was performed in addition to standard fluoroscopy in 51 cases. The volar angular stable plate oesteosyntheses were analyzed intraoperatively and, if necessary, improved immediately. The duration of the scan and radiation exposure dose were measured. On average, performance of the scan and analysis of the CT dataset took 6.7 minutes. In 31.3% of the surgeries a misplacement of screws was detected and correction was done immediately. C-arm CT imaging can easily be integrated in the normal course of surgery. As a complement to the standard 2D-fluoroscopy, the C-arm CT is a useful tool to evaluate the quality of osteosynthesis.

MRI graduation of osseous reaction and drill hole consolidation after arthroscopic Bankart repair with PLLA anchors and the clinical relevance.

PURPOSE: Conventionally, radiography studies revealed prolonged glenoidal drill hole visibilities with an unclear influence to the clinical outcome after arthroscopic Bankart repair using Poly-Laevo-Lactic-Acid (PLLA) anchors. The primary aim of the present study was the separated assessment of drill hole consolidation (DHC) and the concomitant osseous reaction (OR) of the glenoidal bio-degradation process in new specific magnetic resonance grading systems. In accordance with the specific DHC and the OR graduation, the clinical relevance was the secondary focus. METHODS: Twenty-eight patients with arthroscopic Bankart repair using knotless PLLA anchors were prospectively followed and analyzed using a clinical scoring system (3, 6, 15 and 32 months). The T2-weighted OR and T1-weighted DHC were assessed using specific magnetic resonance imaging grading protocols (15 and 32 months). RESULTS: Longitudinal assessments revealed successive clinical status improvements over time (32 months: Rowe 95.7 ± 3.8; Walch-Duplay 93.8 ± 6.6; Constant 93.9 ± 4.5; ASES 93.8 ± 6.9; DASH 28.6 ± 7.2; NAS(pain) 1.1 ± 1.3; NAS(function) 1.3 ± 1.4). The initial OR level regressed over the 15-32 month period while the DHC showed significant drill hole reductions (P < 0.05). The inferior glenoid revealed a significantly increased bio-degradation capacity (P < 0.05) with drill hole enlargements in 14.3%. Neither the OR nor the drill hole enlargements influenced the clinical status. In no case were clinical or radiologic signs for a foreign body reaction. CONCLUSION: Knotless bio-anchors provide secure glenoidal fixation for Bankart repair without any specific clinical or MR evidence of an inflammatory response. The clinical status remained unaffected by the bio-degradation process. LEVEL OF EVIDENCE: IV.

MRI assessment of the structural labrum integrity after Bankart repair using knotless bio-anchors.

PURPOSE: Adequate labral restorations after Bankart repair have been demonstrated in cadaver models for knot-tying and knotless anchors and in vivo by magnetic resonance imaging for knot tying. The influence of glenoidal bio-degradation on clinical outcome, and conclusions regarding drill hole enlargement and foreign body reactions remain controversial. METHODS: The labrum was analyzed in magnetic resonance images for 37 consecutive patients with Bankart repair using knotless PLLA anchors and for 31 volunteers as radiologic controls. The labrum was assessed regarding slope, height index (quotient between labral height to the glenoid height), and labrum morphology in axial and coronal T2 images. Glenoidal bio-degradation was graded in terms of the drill hole configuration in T1 images and the corresponding osseous reaction in T2 images. Constant-Murley, Walch-Duplay, and Rowe scoring were carried out preoperatively and at follow-up. RESULTS: At 15 months after arthroscopy, the anterior slope (24.8°), height index (3.0), inferior slope (25.4°), and height index (2.5) were not significantly different from control values. Morphologic analysis revealed significant changes in the Bankart group (P < 0.05) that were influenced by the number of preoperative dislocations. Bio-degradation proceeded slowly with no evidence of drill hole enlargement. Osseous reactions were significantly greater in inferior compared to superior implants. The clinical scores were excellent and were not influenced by bio-degradation. CONCLUSIONS: Knotless anchors facilitate labral restoration that is comparable to the knot-tying approach. Bio-degradation proceeds slowly without clinical or radiologic evidence of foreign body reactions. LEVEL OF EVIDENCE: III.

Differential deposition of emollients from tripartite formulation systems.

To date, emollients are included in skin care formulations although not much is known about their adsorption/deposition properties and/or the interactions of the constituents within these multi-component systems. The modulation of the adsorption/deposition via the use of specific surfactant and/or emollient systems could therefore help to increase performance and sensorial benefits as well as to reduce adverse effects. In this study, the effects of various tripartite systems consisting of sodium laureth sulphate (SLES), a co-surfactant and an emollient were studied. The two different emollients tested adsorbed with varying amounts although the same surfactant/co-surfactant system was used. Interestingly, the deposition of both SLES and/or the emollient is also substantially influenced by the emollient component itself as well as by the co-surfactant used. Sensory assessments showed that although SLES has a negative effect on the skin feel, adsorbed emollients improve skin softness and smoothness. These results show that optimization of performance is possible when using a co-surfactant best suited for the emollient.

Measurements of the quadriceps femoris function after meniscus refixation at the stable athlete's knee.

INTRODUCTION: The purpose of this study was to monitor the muscular changes regarding the isokinetic strength and torque pattern of the quadriceps femoris at the stable athlete's knee after meniscus tear refixation. MATERIALS AND METHODS: Therefore 15 athletes (10 male, 5 female) performing recreational or competitional sports at least five times a week before injury were retrospectively examined in the average 2.5 years after isolated arthroscopic meniscus refixation using Inside Out technique. Next to function and sport activity focused scores the isokinetic peak torque (PT) and in the EMG have been analyzed compared to the uninjured knee. RESULTS: The mean age was 31.26 years. The time between injury and surgery was in the average 13.7 days. According to our first results the data suggest a complete recovery of functional and muscular pattern after meniscus refixation at the stable athlete's knee. No significant EMG changes for quadriceps femoris were detectable. The PT was fully recovered. The functional and sport activity score analysis (Lysholm and Tegner score) showed no changes in the postoperative long-term follow up compared to the preinjured status. CONCLUSION: Examining isokinetic PT and the EMG of the quadriceps femoris, these data show no side-to-side differences. Regarding the function and sports activity score system, the functionally high demand patients seem to profit by this procedure.

Comparative studies on the ocular and dermal irritation potential of surfactants.

Comparative studies on the irritation potential of 18 surfactants were performed using the same stock solution of surfactant for each study. The ocular irritation potential of surfactants was studied using the red blood cell test (RBC), the hen's egg test-chorioallantoic membrane (HET-CAM) and the Skinethic ocular tissue model. The skin irritation potential was assessed based on data obtained from human studies using a 24h epicutaneous patch test (ECT) and a soap chamber test (SCT). The same pH and active substance (AS) content for all surfactants tested was used depending on the test conducted. In general, clusters of substances with varying irritation potential were identified similarly by most tests. These results show that when using standardized test conditions in which pH and % AS are the same for each surfactant tested, there is a good correlation between the in vitro ocular irritation assays themselves as well as between the dermal and ocular irritation assays. In particular the RBC test seems to be not only highly predictive for ocular irritation (H(50)/DI) but also for dermal irritation and changes in barrier function (DI) induced by surfactants.

Chronobiology: biological clocks and rhythms of the skin.

The cyclicity of time affects virtually all aspects of our being and is the basis of the underlying rhythmicity which is typical of our lives. To 'tell time', most living organisms use internal timing mechanisms known as 'biological clocks'. These 'clocks' coordinate our physiological and behavioral functions and interactions with our environment. One of the strongest influences on rhythmicity is the solar day. The study of these temporal rhythms in biological systems has been coined chronobiology. With the present article we aim to give an overview on chronobiology. Examples of chronobiological effects on skin will be described. Particular emphasis will be placed on circadian rhythms (including rhythms that take place within a 24-hour period, including so-called infradian and/or diurnal rhythms) but also on seasonal variations (circaannual rhythms).

An interlaboratory comparison of methods used to assess antioxidant potentials.

Many analytical methods are used to measure the antioxidative activity of substances yet little is known about the comparability of the test results between laboratories. After an initial evaluation of a broad range of methods conducted by one laboratory, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the trolox equivalent antioxidant capacity (TEAC) assay, the lipid assay (or 2,2'-azobis(2-aminepropane) (ABAP) assay) and the thiobarbituric acid (TBA) assay were selected to be evaluated in the interlaboratory study. The antioxidative potentials of trolox, tocopherol, lipochroman-6, ascorbic acid, 4-methyl-brenzcatechin, and/or 3,5-di-tert-butyl-4-hydroxytoluene (BHT) were assessed using each of the methods. These methods were then evaluated in respect of their reproducibility and classification properties. Based on the results of this study, the DPPH assay followed by the TEAC assay yielded the best results based on reproducibility and sensitivity both within one laboratory and between laboratories. The results of the interlaboratory study were then compared with the single center results obtained from the commercially available photochemolumiescence (PCL) kit. To assess the transferability of chemical data to biological systems, they were also compared with the single center results obtained using the cell-based Dichlorodihydrofluoresceine (DCFH) assay.

Overexpression of CD40 ligand in murine epidermis results in chronic skin inflammation and systemic autoimmunity.

CD40-CD40 ligand (L) interactions play a pivotal role in immune-mediated inflammatory responses via the activation of antigen-presenting cells (APCs). To investigate the effects of continuous activation of resident tissue APCs, in this case the Langerhans cells (LCs) of the skin, CD40L expression was targeted to the basal keratinocytes of the epidermis of mice using the keratin-14 promoter. Approximately 80% of the transgenic (Tg) mice spontaneously developed dermatitis on the ears, face, tail, and/or paws. Compared with littermates, Tgs had a >90% decrease in epidermal LCs yet increased numbers within the dermis suggestive of enhanced emigration of CD40-activated LCs. Tgs also displayed massive regional lymphadenopathy with increased numbers of dendritic cells and B cells. Moreover, a decrease in IgM and an increase in IgG1/IgG2a/IgG2b/IgE serum concentrations was detectable. Screening for autoantibodies revealed the presence of antinuclear antibodies and anti-dsDNA antibodies implicative of systemic autoimmunity. Accordingly, renal Ig deposits, proteinuria, and lung fibrosis were observed. Adoptive transfer of T cells from Tgs to nonTg recipients evoked the development of skin lesions similar to those found in the Tgs. Dermatitis also developed in B cell-deficient CD40L Tg mice. These findings suggest that in situ activation of LCs by CD40L in the skin not only leads to chronic inflammatory dermatitis but also to systemic mixed-connective-tissue-like autoimmune disorders, possibly by breaking immune tolerance against the skin.

Mycophenolate mofetil impairs the maturation and function of murine dendritic cells.

The immunosuppressive drug, mycophenolate mofetil (MMF), has been successfully introduced in allogeneic transplantation medicine and, more recently, in the treatment of autoimmune skin disorders. MMF inhibits lymphocyte proliferation via a blockade of the enzyme inosine 5'-monophosphate dehydrogenase, an enzyme on which lymphocytes solely depend to generate the purines necessary for DNA/RNA synthesis. To investigate the effects of MMF on cutaneous immune responses, a murine model of contact hypersensitivity (CHS) was used, with oxazolone or trinitrochlorobenzene as a contact allergen. Compared with the respective vehicle, i.p. applied MMF significantly inhibited the elicitation and, surprisingly, the induction of CHS responses. This prompted further studies into the effects of MMF on Ag presentation. Bone marrow-derived dendritic cells (DC) were cultured with GM-CSF and IL-4 in the presence of MMF and were tested for their Ag-presenting capacity. Sensitization and elicitation of CHS and delayed-type hypersensitivity responses by s. c. injected haptenated DC were reduced upon preincubation of DC with MMF. CHS responses were not impaired upon resensitization, indicating that MMF does not induce hapten-specific immunotolerance. In addition, MMF decreased the ability of DC to stimulate allogeneic T cells in MLR assays. Accordingly, flow cytometric analyses revealed a dose-dependent reduction of the expression of CD40, CD80, CD86, I-A, and ICAM-1 on DC with a concurrent reduction of IL-12 production. These data suggest that MMF, in addition to affecting T lymphocytes, directly affects APC, resulting in an impairment of immune responses. They furthermore point to a possible role of inosine 5'-monophosphate dehydrogenase in the maturation of DC.

Reduced ultraviolet-induced carcinogenesis in mice with a functional disruption in B7-mediated costimulation.

Immunosuppression by UV light contributes significantly to the induction of skin cancer by suppressing the cell-mediated immune responses which control the development of carcinogenesis. The B7/CD28-CTLA-4 signaling pathway provides costimulatory signals essential for Ag-specific T cell activation. To investigate the role of this pathway in photocarcinogenesis, we utilized transgenic (Tg) mice which constitutively express CTLA-4Ig, a high-affinity CD28/CTLA-4 antagonist that binds to both B7-1 and B7-2. The transgene is driven by a skin-specific promoter yielding high levels of CTLA-4Ig in the skin and serum. Chronic UV exposure of CTLA-4Ig Tg mice resulted in significantly reduced numbers of skin tumors, when compared to control mice. In addition, Tg mice were resistant to UV-induced suppression of delayed-type hypersensitivity responses to alloantigens. Most importantly, upon stimulation with mitogens and alloantigens, T cells isolated from CTLA-4Ig Tg mice produced significantly less IL-4 but more IFN-gamma compared to control T cells, suggesting an impaired Th2 response and a relative increase of Th1-type immunity. Together, these data show that overall B7 engagement directs immune responses toward the Th2 pathway. Moreover, they point out the crucial role of Th1 immune reactions in the protection against photocarcinogenesis.

Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.

Bone marrow-derived dendritic cells (BmDC) are potent APC and can promote antitumor immunity in mice when pulsed with tumor Ag. This study aimed to define the culture conditions and maturation stages of BmDC that enable them to optimally function as APC in vivo. BmDC cultured under various conditions (granulocyte-macrophage CSF (GM-CSF) or GM-CSF plus IL-4 alone or in combination with Flt3 ligand, TNF-alpha, LPS, or CD40 ligand (CD40L)) were analyzed morphologically, phenotypically, and functionally and were tested for their ability to promote prophylactic and/or therapeutic antitumor immunity. Each of the culture conditions generated typical BmDC. Whereas cells cultured in GM-CSF alone were functionally immature, cells incubated with CD40L or LPS were mature BmDC, as evident by morphology, capacity to internalize Ag, migration into regional lymph nodes, IL-12 secretion, and alloantigen or peptide Ag presentation in vitro. The remaining cultures exhibited intermediate dendritic cell maturation. The in vivo Ag-presenting capacity of BmDC was compared with respect to induction of both protective tumor immunity and immunotherapy of established tumors, using the poorly immunogenic squamous cell carcinoma, KLN205. In correspondence to their maturation stage, BmDC cultured in the presence of CD40L exhibited the most potent immunostimulatory effects. In general, although not entirely, the capacity of BmDC to induce an antitumor immune response in vivo correlated to their degree of maturation. The present data support the clinical use of mature, rather than immature, tumor Ag-pulsed dendritic cells as cancer vaccines and identifies CD40L as a potent stimulus to enhance their in vivo Ag-presenting capacity.

Impaired cutaneous immune responses in Thy-1-deficient mice.

Thy-1 is a cell surface glycoprotein expressed mainly on brain and lymphoid tissue. Although the functions of Thy-1 are incompletely understood, evidence exists that Thy-1 participates in T cell activation. To examine the functional role of Thy-1 in cutaneous immune responses in vivo, Thy-1 gene-targeted mice (Thy-1-/-) and wild-type mice (Thy-1+/+) were immunized with the hapten oxazolone. After challenge with oxazolone, contact hypersensitivity responses in Thy-1-/- mice were reduced by 25% compared with Thy-1+/+ mice. Likewise, irritant dermatitis induced by croton oil was also decreased. In addition, Thy-1-/- mice showed a significantly reduced delayed-type hypersensitivity response after injection of allogeneic spleen cells into the hind footpads of allosensitized animals when compared with Thy-1+/+ mice. Moreover, proliferative responses to immobilized anti-CD3 were decreased in peripheral Thy-1-/- lymphocytes; this decrease was associated with a significantly reduced intracellular Ca2+ influx and protein tyrosine phosphorylation, indicating impairment of early lymphocyte activation. In contrast, the T cell proliferation induced by mitogens was normal, suggesting that Thy-1 expression weakly contributes to TCR-mediated T cell activation. Epidermal Langerhans cells and bone marrow-derived dendritic cells from Thy-1-/- mice exhibited a normal expression of costimulatory surface molecules as well as an unaltered ability to stimulate allogeneic T cells. Taken together, these findings demonstrate that a lack of Thy-1 expression does not generally compromise the immune system; however, Thy-1 expression may be involved in the fine-tuning of T cell-mediated immune responses.