SID Posters 2016

Posted on May 2, 2016 |
Categories Meetings, Posters, Society for Investigative Dermatology

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Induction of an Atopic Dermatitis-Like Phenotype in a Full-Thickness in vitro Human Skin Model 
Abstract #356 | Poster Session II, Saturday, May 14, 2016 10:00 am – 12:00 pm
Michael Bachelor, Jennifer Molignano, Jonathan Oldach, Gina Stolper, Max  Li, Alex Armento and Patrick Hayden

Atopic dermatitis (AD) is the most common inflammatory disease of the skin characterized by defects in keratinocyte differentiation and skin barrier dysfunction.  AD is highly prevalent, affecting 15-30% of children and 2-10% of adults, or 20% of the population worldwide.  Despite its prevalence, a highly differentiated in vitro model of AD is not currently commercially available to facilitate basic research and therapeutic development.  In addition, the few animal models that exist do not adequately recapitulate the human AD phenotype.  We utilized the EpiDermFT (EFT-400) in vitro skin model to evaluate the effects of the pro-inflammatory Th1 cytokine TNF-alpha and Th2 cytokines (IL-4, IL-13, IL-25, and IL-31) on markers of differentiation and skin barrier in order to recapitulate an AD-like phenotype.  Histological analysis revealed treatment with IL-4, IL-13, IL-25, and IL-31 reduced the presence of the stratum granulosum and induced spongiosis to varying degrees in the epidermis.  Gene expression analysis also revealed changes consistent with those found in AD.  More specifically, treatment with IL-4, IL-13, IL-25, IL-31 and or TNF-alpha induced significant increases in the mRNA expression of thymic stromal lymphoprotein (TSLP) and thymus and activation regulated chemokine (TARC/CCL17) and decreases in both filaggrin and loricrin.  Treatment with Th2 cytokines also increased secretion of the pro-inflammatory cytokine, IL-8.  Reduced filaggrin expression was confirmed at the protein level by immunohistochemical analysis.  These findings demonstrate that supplementation with Th1 (TNF-a) and Th2 cytokines induce a phenotype in the EpiDermFT tissue model consistent with the hallmarks of AD, including altered differentiation and barrier function.  The model described here may therefore be useful for evaluating the effects of active ingredients and other molecules used in the treatment of atopic dermatitis.

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Evaluation of Wound Healing in a Full-Thickness in vitro Human Skin Model
Abstract #721 | Poster Session I, Thursday, May 12, 2016 10:15 am – 12:15 pm
Michael Bachelor, Jonathan Oldach, Gina Stolper, Max Li, Alex Armento and Patrick Hayden

Wound healing is a fundamental process to re-establish tissue integrity and skin barrier function.   A model of wound healing was created by introducing epidermal wounds in a full thickness in vitro human skin model (EpiDermFT) using a 3-mm punch biopsy and subsequently evaluated at multiple recovery time points.  EpiDermFT exhibits stratified epidermal components and a fully developed basement membrane resembling in vivo skin in regard to both morphology and barrier function.  Historically, EpiDermFT has been used to evaluate re-epithelization of the wound by: a) manually bisecting the tissues through the center of the wound, b) staining with hematoxylin and eosin, and c) quantifying migration from the wound origin.  Accurate bisection of the wound is difficult and often leads to variability in assay results.  Here we describe a novel method of visualizing wound re-epithelization in situ simplifying analysis and reducing introduction of variables inherent in tissue processing that could potentially confound data.  Following wounding, tissues were fixed and immunostained with markers of epidermal differentiation as well as a marker of fibroblasts allowing simultaneous visualization of migrating keratinocytes (keratin 14), differentiated suprabasal cells (involucrin), and dermal fibroblasts (vimentin) within the wound.  Histological and immunohistochemical analysis showed keratinocyte migration at 2 days following wounding.  In both methods, wounded tissues cultured without growth factors (2% human serum) had a reduced healing rate in which keratinocytes did not cover the entire wound within a 6 day timeframe.  In contrast, wounded tissues cultured with growth factors demonstrated a dramatic increase in healing rate as keratinocyte migration completely covered the wounded area by day 6.  In conclusion, this novel method of evaluating re-epithelization by utilizing immunohistochemical markers of differentiation is a quicker and more reproducible method of analyzing wound healing.

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Measurement of Skin Pigmentation Using a Chromameter in a 3-Dimensional Epidermal Model Containing Functional Melanocytes
Abstract #607 | Poster Session I, Thursday, May 12, 2016 10:15 am – 12:15 pm
Michael Bachelor, Bridget Breyfogle, Alex Armento and Mitchell Klausner

Various cosmetic or skin care pharmaceutical formulations augment skin pigmentation either for the intended purpose of skin lightening or as a side effect induced by medication.   A convenient way to screen such effects utilizes the MelanoDerm tissue model, a highly differentiated, three-dimensional tissue culture model of human epidermis containing normal human melanocytes and keratinocytes.  Use of this model can provide valuable in vitro data as an early screening tool prior to the commencement of costly clinical trials.  In this study, pigmentation was evaluated over the course of 2-3 weeks using a tristimulus chromometer to measure brightness (L*), yellowness (b*) and redness (a*) in MelanoDerm tissue produced with normal human melanocytes from Black, Asian, or Caucasian donors.  In parallel to measurements taken with the chromameter, total melanin content of tissues was also quantified.  Over time, cultures became increasingly pigmented with retention of normal epithelial morphology with the expected pigmentation level of the donor tissue, i.e. Black>Asian>Caucasian when cultured in media containing alpha-MSH and beta-FGF.  Several over-the-counter skin lightening products were also evaluated in cultures containing normal human melanocytes from Black donors. Over the 2-3 week treatment period, control cultures became increasingly pigmented while tissues treated topically with cosmetic skin lightening agents containing tyosinase inhibitors such as kojic acid and magnesium ascorbyl phosphate remained distinctly lighter when compared to control cultures.  After 14 days in culture, total melanin content was found to inversely correlate with surface reflectance (L*).  The results described herein suggest that this model is useful for evaluating effects on melanogenesis, skin lightening, and other pigmentation phenomena of skin in vitro.  In particular, this study highlights two distinct endpoints, total melanin content and skin color measurement that can be used to evaluate skin pigmentation in vitro.

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Targeting the IL-17 – CCL20 Pathway to Screen Drug Candidates in an Organotypic Psoriasis Tissue Model  
Abstract #357 | Poster Session I, Thursday, May 12, 2016 10:15 am – 12:15 pm
Seyoum Ayehunie, Tim Landry, Zachary Stevens, Alex Armento, Patrick Hayden, and Mitchell Klausner

Psoriasis is a chronic inflammatory skin disease marked by hyperproliferation, abnormal keratinocyte (KC) differentiation, and leukocyte infiltration.  Research into the pathogenesis of psoriasis has been hampered by the lack of in vitro human cell based tissue or animal models that accurately mimic the biology of psoriatic phenotypes. Since T cells, particularly Th17 cells (IL-17 producing cells), are implicated in inflammatory skin diseases and because targeting IL-17 has been a promising approach in clearing moderate to severe plaque psoriasis, we investigated the role of IL-17 alone or in combination with IFN-γ (Th1 cytokines) in exacerbating inflammatory responses using a reconstructed 3D human psoriatic tissue model.  Normal human keratinocytes (KC) and psoriatic fibroblasts from lesional and non-lesional sites were harvested from primary tissue and cultured using defined serum-free medium to form a highly differentiated 3-dimensional tissue model. The reconstructed psoriatic tissue model was exposed to IL-17 / IFN-γ for up to 96 hr (4X exposure). The results showed that: 1) IL-17 increases the Th-17 cell chemoattractant (CCL-20), the neutrophil chemoatractant (IL-8), and the anti microbial peptides (HBD2 and elafin (PI3) by 6.9, 2.8, 2.4, and 3.4 fold, respectively.  2) IFN-γ induces overexpression of the chemoattractant for activated T cells (CXCL11), IL-8, and TNF-α by 324, 3.7, and 2.2 fold, respectively.  3). IL-17 and IFN-γ exacerbate inflammation synergistically by increasing expression levels of the neutrophil chemoattractants (CXCL5, IL-8), HBD2, PI3, and TNF-α by > 3 fold. Here, we show that a self-sustaining inflammatory feedback loop that involves the T-cell cytokines (IL-17, IFN- γ) and KC/fibroblast derived CCL20, CXCL5, HBD2, PI3, and IL-8 is established.  Release/expression level of CCL-20, IL-8, IL-6, and CXCL-5 can serve as surrogate markers to examine effect of anti-IL17 drugs.  In conclusion, these surrogate markers can be used as valuable tools to screen candidate therapeutics for psoriasis.

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Late-Breaking Posters

Determination of Contact Sensitization Potential of Chemicals Using in vitro Reconstructed Normal Human Epidermal Model EpiDerm: Impact of the Modality of Application
Poster ID: LB756  | Poster Session II, Saturday, May 14, 2016 10:00 am – 12:00 pm
Letasiova S.1, Galbiati V.2, Kandarova H.1, Corsini E.2 , Lehmeier D.3, Gehrke H.3  1 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, 2 Laboratory of Toxicology, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy, 3 Eurofins BioPharma Product Testing Munich GmbH, Germany

Assessment of skin sensitization potential has traditionally been conducted in animal models, such as the Mouse Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). However, a growing focus and consensus for minimizing animal use have stimulated the development of in vitro methods to assess skin sensitization. Interleukin-18 (IL-18) release in reconstructed human epidermal models has been identified as a potentially useful endpoint for the identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (1). The purpose of this study was to investigate the impact of the modality of chemical exposure on the predictive capacity of the assay. EpiDerm tissue viability assessed by MTT assay and IL-18 release assessed by ELISA were evaluated after 24 h topical exposure to test chemicals either impregnated in 8 mm diameter paper filters or directly applied to the surface of EpiDerm. Acetone: olive oil (4:1) was used as vehicle in all cases. A total of five chemicals from 3 different sources were tested. The testing set included 3 senzitizers, namely 2,4-dinitrochlorobenzene, cinnamaldehyde and isoeugenol/eugenol, and 2 non senzitizers, lactic acid and salicylic acid. Four independent dose – response experiments were conducted in 3 laboratories, resulting in correct prediction of the sensitizing potency of test chemicals. The assessment of IL-18 release using in vitro reconstructed normal human epidermal model EpiDerm appears to be a promising tool for in vitro determination of contact sensitization potential.

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Development, Optimization and Validation of an in Vitro Skin Irritation Test for Medical Devices Using the Reconstructed Human Tissue Model EpiDerm 
Poster ID: LB794  | Poster Session II, Saturday, May 14, 2016 10:00 am – 12:00 pm
Kandarova, Helena1, 4; Willoughby, Jamin A., Sr.2; de Jong, Wim H.3; Bachelor, Michael A.4; Letasiova, Silvia1; Milasova, Tatiana1; Breyfogle, Bridget4; de la Fonteyne, Liset3; Haishima, Yuji5; Coleman, Kelly P.1 MatTek IVLSL, Bratislava, Slovakia, 2 Cyprotex US LLC, Kalamazoo, MI, United States, 3 RIVM, Bilthoven, Netherlands, 4 MatTek Corporation, Ashland, MA, United States, 5 Division of Medical Devices – NIHS, Tokyo, Japan, 6 Medtronic, PLC, Minneapolis, MN, United States

Assessment of dermal irritation is an essential component of the safety evaluation of medical devices.  Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals (OECD TG 439).  However, medical device extracts are dilute solutions with low irritation potential, therefore validated RhE-methods needed to be modified to reflect the needs of ISO 10993.  A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymer extracts (Casas et al., TIV, 2013).  In 2014 a second laboratory assessed the transferability of the assay.  Two additional exposure times were tested along with other medical device materials.  After the successful transfer and standardization of the protocol, nine EU and USA laboratories were trained in the use of the protocol in preparation for a validation study.  All laboratories produced data that was nearly 100% in agreement with predictions for the selected references.  Two of the laboratories performed additional tests with heat-pressed PVC sheets spiked with Genapol X-080 (Y-4 polymer), Vicryl suture, and polymers spiked with heptanoic acid and sodium dodecyl sulfate.  All materials were extracted for 24 or 72 hours in both saline and sesame oil at 37°C.  Significant irritation responses were detected for Y-4 under all conditions.  These results were consistent with those reported by other research groups involved in the upcoming validation study.  Vicryl suture was negative and spiked polymers were either positive or negative depending on the extraction solvent.  Therefore, we conclude that a modified RhE skin irritation assay has the ability to address the skin irritation potential of medical devices, however, standardization and focus on technical issues is essential for accurate prediction.  A round robin validation study of in vitro skin irritation testing for assessment of medical device extracts is beginning in March 2016.

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