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LB997 Inducers of GILT expression in human melanoma
ABSTRACTS | LB996
LB997
A newly developed pigmentation correcting serum (LYT2) targeting multiple pathway of melanogenesis K Kadoya, R Chung, P Lai, A Nguyen and RC Mehta Allergan, Irvine, CA Hyperpigmentation of the skin is an important condition which is difficult to treat. It is caused by many factors such as sun exposure, aging, inflammation and genetic background. Correction of hyperpigmentary condition requires control of all stages of melanin lifecycle including melanocyte activation, melanosome development, melanin production, melanin distribution and epidermal turnover. We have developed a new product with a combination of ingredients that can modulate these key pathways. The goal of the study was to evaluate the effect of pigmentation correcting serum (LYT2) on various pathways involved in the melanin lifecycle. MEL-300-B (MatTek) constructed skin tissues were used for this study. Samples were collected after two weeks and evaluated for melanin quantification, gene expression of key targets and histological assessments. LYT2-treated tissues showed a reduction in melanin content by 45%7.65% compared to control. The expression of proopiomelanocortin (POMC) and microphthalmia transcription factor (MITF) genes which are involved with melanocyte activation showed down-regulation by 67.7% and 53.9%, respectively. Key genes of melanosome development, Melan-A, showed a 39.9% down-regulation. Melanin synthesis genes tyrosinase (TYR), tyrosinase related protein 1 (TYRP1) and TYRP2 also showed down-regulation of 53.4%, 57.5% and 34.8% respectively. Melanosome transfer inhibitory genes dickkopf-related pretein-1 (DKK-1) showed a 1236% upregulation. Histological analysis with Fontana Masson staining showed reduction of melanin particles compared to controls. These results indicate that newly developed pigmentation correcting serum (LYT2) is targeting multiple key pathways to control hyperpigmentation in the skin.
Inducers of GILT expression in human melanoma H Menon1, L Meador1, H Cui2, D Roe3, D DiCaudo4 and K Hastings1 1 University of Arizona College of Medicine, Phoenix, AZ, 2 University of Arizona Cancer Center, Tucson, AZ, 3 University of Arizona Cancer Center, University of Arizona Mel and Enid Zuckerman College of Public Health, Tucson, AZ and 4 Mayo Clinic, Scottsdale, AZ T cell-mediated immunity has the ability to produce durable anti-melanoma responses resulting in improved survival of patients with advanced melanoma. Gamma-interferoninducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells and facilitates cross-presentation on MHC class I for activation of CD8+ T cells. Our prior study found that GILT protein expression is increased in tumor cells, tumor-infiltrating antigen presenting cells and keratinocytes in melanoma specimens compared to nevi. We compared GILT expression in halo nevi specimens to nevi without lymphocytic infiltrate by immunohistochemistry. GILT expression was increased in the three cell types in halo nevi compared to nevi (p < 0.05 for each), suggesting that GILT expression is induced by the inflammatory environment and is not specific for malignancy. Analysis of a publicly available gene expression profiling cohort of 457 cutaneous melanoma patients revealed that GILT mRNA expression was associated with IFN-g (p < 0.0001) and TNF expression (p < 0.0001), but not IL-1b expression. In vitro exposure to IFN-g induced GILT protein expression in a melanoma cell line, which lacked GILT expression at baseline; we are testing the ability of TNF to induce GILT expression. Together these data support that inflammatory cytokines induce GILT expression in human melanoma.
LB998
LB999
Follicular regeneration in response to wounding in ccca N Clemetson1, GA Okoye2, H Pasieka1, M Oboite1, S Leung1, J Mensah1, S Alessi1, H Hao3, S Kang1 and LA Garza1 1 Dermatology, Johns Hopkins Univ, Baltimore, MD, 2 Department of Dermatology, Johns Hopkins University School of Medicine and Bayview Medical Center, Baltimore, MD and 3 Johns Hopkins Univ, Baltimore, MD Central centrifugal cicatricial alopecia (CCCA) is an inflammatory condition that causes permanent scalp alopecia in black women. Treatment options are limited. Wounding has been shown to generate hair follicles de novo, likely under the influence of Wnt signaling. Ablative carbon dioxide (CO2) laser therapy creates small wounds in the skin. In this exploratory study, we hypothesized that follicular regeneration in CCCA may be demonstrated by increased scalp hair counts, in response to wounding with a CO2 laser. We enrolled 17 women with CCCA, age 18+ years and treated two areas of the scalp with a fractionated CO2 laser. Pulse energy was 55mJ, 110mJ or 240mJ. Each subject was her own control, using a split scalp model. Photographs and punch biopsies were obtained. The difference in hair counts (from baseline to final visit) was calculated and compared using the Wilcoxon signedrank test. From the baseline to final visits, average hair counts increased 73% vs 48% in peripherally treated vs untreated areas. Hair counts increased 66% vs 49% in centrally treated vs untreated areas. At 240mJ, the increase in hair counts in treated vs untreated peripheral areas was significant (p¼0.03). Centrally, there was no significant difference at all pulse energy levels. While the general trend was toward an increase in hair counts in all areas, the most significant change was noted at peripheral sites treated at the highest energy setting. These findings support our hypothesis that follicular regeneration may occur in CCCA in response to wounding.
Role of transcription factor Ovol2 in skin epithelial regeneration and repair D Haensel, P Sun, A MacLean, Y Zhou, M McNeil, Q Nie and X Dai University of California, Irvine, Irvine, CA Epithelial cells possess the ability to become mesenchymal cells under appropriate conditions. Complete epithelial-to-mesenchymal transition (EMT) generates crucial cell types such as mesoderm and neural crest during embryogenesis, whereas partial EMT underlies pathological processes such as wound healing and cancer metastasis. Whether and how EMT impacts the self-renewal and differentiation of adult epithelial tissue stem cells is not clear. We had previously identified zinc finger transcription factors Ovol1 and Ovol2 as critical inhibitors of EMT during skin and mammary epithelial morphogenesis. Here we investigate the role of Ovol2 in adult skin epithelial regeneration and repair, and its possible connection to EMT regulation. We show that Ovol2 is expressed in adult epidermal basal and hair follicle bulge cells as well as their early progenies. K14-Cre-mediated deletion of Ovol2 delays hair follicle anagen progression and epidermal wound healing. Ovol2-deficient adult epidermal cells show reduced hair regenerating capacity upon transplantation, and display aberrant dissemination behaviors when they migrate to repair cutaneous wounds. FACS analysis reveals a decreased number of hair follicle stem cells (HFSCs) in skin from adult mice lacking Ovol2, and gene expression analysis reveals enhanced EMT and de-enhanced cell cycle gene signatures in Ovol2-deficient keratinocytes. Without Ovol2, HFSCs display mesenchymallike morphology, defective clonal growth, and altered cell division behavior accompanied by an apparent cell cycle arrest between G2/M and G1. Ongoing experiments address the potential molecular mechanisms underlying these regeneration/repair and cellular defects, and data will be presented.
LB1000
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Differentiation of dermal papilla cells into a myogenic lineage for the treatment of duchenne muscular dystrophy M Rashidi1, N Rufaut2, L Jones3, T Partridge4 and R Sinclair5 1 The George Washington University, McLean, VA, 2 University of Melbourne, Dunedin, New Zealand, 3 University of Melbourne, Victoria, Australia, 4 The George Washington University, Washington, DC and 5 University of Melbourne, Melbourne, Victoria, Australia Duchenne muscular dystrophy (DMD) is the commonest muscular dystrophy caused by the absence of dystrophin. Stem cell therapy in DMD is one of the promising approaches for treatment. Multipotent stem cells residing in the hair follicle papilla are highly plastic. We showed that dermal papilla cells (DPC) undergo myogenic differentiation when co-cultured with different types of myoblasts including dystrophic human myoblasts. DPC incorporated into myotubes and upregulated the muscle marker, myogenin, in the co-culture with human myoblasts. DPC incorporation efficiency was low (< 5%) in all co-cultures and differed significantly (p value 0.001) between various types of myoblasts; however, no significant difference was observed between normal and dystrophic human myoblasts (p value 0.001). These encouraging findings suggested that the altered properties of dystrophic human myoblasts did not compromise the myogenic differentiation of DPC in vitro, supporting their in vivo application and possible therapeutic potential. The in vitro effects of galectin-1 and activation of Shh signaling pathway via recombinant Shh (rShh) and purmorphamine, on the myogenic differentiation of DPC, was also evaluated. None of the treatments increased myogenin expression in DPC; but, triggering Shh signaling produced a dose dependent pattern whereby the lower levels of signaling promoted myogenic differentiation while the higher levels inhibited it. Activating Shh signaling upstream of Smo, via purmorphamine, induced a biphasic differentiative response; however, the application of rShh hindered the differentiation of both cell types. Thus, murine DPC are a readily accessible source of stem cells that can undergo myogenic differentiation in vitro and their myogenic differentiation can be enhanced with proper treatment .
B12 Journal of Investigative Dermatology (2017), Volume 137
In vitro examination of Mexican hair shaft morphology using optical coherence tomography M Perper1, M Martinez2, A Maddy1, J Cervantes1, A Eber1, S Verne1, N Vazquez3, K Nouri1 and A Tosti1 1 University of Miami Miller School of Medicine, Miami, FL, 2 University of Miami Miller School of Medicine, Mexico City, Mexico and 3 University of Miami Miller School of Medicine, Monterrey, Mexico Hair diameter, shape, mechanical properties, combability, and moisture content have been previously examined, however primarily in Caucasian, African, and Asian individuals. We describe the distinct cross-sectional and morphological hair characteristics of Hispanics, an ethnic group accounting for 16% of total hair product sales in the United States, yet one that is not sufficiently studied. The VivoSightÒ swept-source multi-beam optical coherence tomography (OCT) system was used to evaluate hair diameter and shape in 30 female volunteers aged 18 to 55 recruited from Hospital Me´dica Sur in Mexico. Three hair samples from each volunteer were measured transversely along three distances, generating 9 crosssectional images, 2 measurements per image, and 18 measurements per patient for a total of 540 diameter measurements in 30 patients. Samples were measured 10 mm from the proximal portion of the fiber, middle position (equidistant point between the root and end), and 10 mm from the distal portion of the fiber. The minimum hair diameter of all measurements (n ¼ 540) was 0.06 mm while the maximum was 0.14 mm. The mean diameter of the hair samples was 0.10 0.01 mm. Of note, we found that Mexican hair resembles Caucasian and Asian hair, in that hair shafts tend to be round-shaped with homogenous diameters throughout the fiber, rather than elliptical, flattened, and irregular, as seen with Afro-ethnic hair. Mexican hair is similar to Asian hair in terms of diameter and shape, and thicker on average than Caucasian hair. Our findings suggest that OCT may be a viable method of hair examination in addition to more traditional approaches such as light, confocal, scanning electron, and transmission electron microscopy. Retailers may use information garnered from this study to tailor hair products to the unique hair characteristics of Hispanics, an ethnic group driving sales in the hair care market.
LB997
A newly developed pigmentation correcting serum (LYT2) targeting multiple pathway of melanogenesis K Kadoya, R Chung, P Lai, A Nguyen and RC Mehta Allergan, Irvine, CA Hyperpigmentation of the skin is an important condition which is difficult to treat. It is caused by many factors such as sun exposure, aging, inflammation and genetic background. Correction of hyperpigmentary condition requires control of all stages of melanin lifecycle including melanocyte activation, melanosome development, melanin production, melanin distribution and epidermal turnover. We have developed a new product with a combination of ingredients that can modulate these key pathways. The goal of the study was to evaluate the effect of pigmentation correcting serum (LYT2) on various pathways involved in the melanin lifecycle. MEL-300-B (MatTek) constructed skin tissues were used for this study. Samples were collected after two weeks and evaluated for melanin quantification, gene expression of key targets and histological assessments. LYT2-treated tissues showed a reduction in melanin content by 45%7.65% compared to control. The expression of proopiomelanocortin (POMC) and microphthalmia transcription factor (MITF) genes which are involved with melanocyte activation showed down-regulation by 67.7% and 53.9%, respectively. Key genes of melanosome development, Melan-A, showed a 39.9% down-regulation. Melanin synthesis genes tyrosinase (TYR), tyrosinase related protein 1 (TYRP1) and TYRP2 also showed down-regulation of 53.4%, 57.5% and 34.8% respectively. Melanosome transfer inhibitory genes dickkopf-related pretein-1 (DKK-1) showed a 1236% upregulation. Histological analysis with Fontana Masson staining showed reduction of melanin particles compared to controls. These results indicate that newly developed pigmentation correcting serum (LYT2) is targeting multiple key pathways to control hyperpigmentation in the skin.
Inducers of GILT expression in human melanoma H Menon1, L Meador1, H Cui2, D Roe3, D DiCaudo4 and K Hastings1 1 University of Arizona College of Medicine, Phoenix, AZ, 2 University of Arizona Cancer Center, Tucson, AZ, 3 University of Arizona Cancer Center, University of Arizona Mel and Enid Zuckerman College of Public Health, Tucson, AZ and 4 Mayo Clinic, Scottsdale, AZ T cell-mediated immunity has the ability to produce durable anti-melanoma responses resulting in improved survival of patients with advanced melanoma. Gamma-interferoninducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells and facilitates cross-presentation on MHC class I for activation of CD8+ T cells. Our prior study found that GILT protein expression is increased in tumor cells, tumor-infiltrating antigen presenting cells and keratinocytes in melanoma specimens compared to nevi. We compared GILT expression in halo nevi specimens to nevi without lymphocytic infiltrate by immunohistochemistry. GILT expression was increased in the three cell types in halo nevi compared to nevi (p < 0.05 for each), suggesting that GILT expression is induced by the inflammatory environment and is not specific for malignancy. Analysis of a publicly available gene expression profiling cohort of 457 cutaneous melanoma patients revealed that GILT mRNA expression was associated with IFN-g (p < 0.0001) and TNF expression (p < 0.0001), but not IL-1b expression. In vitro exposure to IFN-g induced GILT protein expression in a melanoma cell line, which lacked GILT expression at baseline; we are testing the ability of TNF to induce GILT expression. Together these data support that inflammatory cytokines induce GILT expression in human melanoma.
LB998
LB999
Follicular regeneration in response to wounding in ccca N Clemetson1, GA Okoye2, H Pasieka1, M Oboite1, S Leung1, J Mensah1, S Alessi1, H Hao3, S Kang1 and LA Garza1 1 Dermatology, Johns Hopkins Univ, Baltimore, MD, 2 Department of Dermatology, Johns Hopkins University School of Medicine and Bayview Medical Center, Baltimore, MD and 3 Johns Hopkins Univ, Baltimore, MD Central centrifugal cicatricial alopecia (CCCA) is an inflammatory condition that causes permanent scalp alopecia in black women. Treatment options are limited. Wounding has been shown to generate hair follicles de novo, likely under the influence of Wnt signaling. Ablative carbon dioxide (CO2) laser therapy creates small wounds in the skin. In this exploratory study, we hypothesized that follicular regeneration in CCCA may be demonstrated by increased scalp hair counts, in response to wounding with a CO2 laser. We enrolled 17 women with CCCA, age 18+ years and treated two areas of the scalp with a fractionated CO2 laser. Pulse energy was 55mJ, 110mJ or 240mJ. Each subject was her own control, using a split scalp model. Photographs and punch biopsies were obtained. The difference in hair counts (from baseline to final visit) was calculated and compared using the Wilcoxon signedrank test. From the baseline to final visits, average hair counts increased 73% vs 48% in peripherally treated vs untreated areas. Hair counts increased 66% vs 49% in centrally treated vs untreated areas. At 240mJ, the increase in hair counts in treated vs untreated peripheral areas was significant (p¼0.03). Centrally, there was no significant difference at all pulse energy levels. While the general trend was toward an increase in hair counts in all areas, the most significant change was noted at peripheral sites treated at the highest energy setting. These findings support our hypothesis that follicular regeneration may occur in CCCA in response to wounding.
Role of transcription factor Ovol2 in skin epithelial regeneration and repair D Haensel, P Sun, A MacLean, Y Zhou, M McNeil, Q Nie and X Dai University of California, Irvine, Irvine, CA Epithelial cells possess the ability to become mesenchymal cells under appropriate conditions. Complete epithelial-to-mesenchymal transition (EMT) generates crucial cell types such as mesoderm and neural crest during embryogenesis, whereas partial EMT underlies pathological processes such as wound healing and cancer metastasis. Whether and how EMT impacts the self-renewal and differentiation of adult epithelial tissue stem cells is not clear. We had previously identified zinc finger transcription factors Ovol1 and Ovol2 as critical inhibitors of EMT during skin and mammary epithelial morphogenesis. Here we investigate the role of Ovol2 in adult skin epithelial regeneration and repair, and its possible connection to EMT regulation. We show that Ovol2 is expressed in adult epidermal basal and hair follicle bulge cells as well as their early progenies. K14-Cre-mediated deletion of Ovol2 delays hair follicle anagen progression and epidermal wound healing. Ovol2-deficient adult epidermal cells show reduced hair regenerating capacity upon transplantation, and display aberrant dissemination behaviors when they migrate to repair cutaneous wounds. FACS analysis reveals a decreased number of hair follicle stem cells (HFSCs) in skin from adult mice lacking Ovol2, and gene expression analysis reveals enhanced EMT and de-enhanced cell cycle gene signatures in Ovol2-deficient keratinocytes. Without Ovol2, HFSCs display mesenchymallike morphology, defective clonal growth, and altered cell division behavior accompanied by an apparent cell cycle arrest between G2/M and G1. Ongoing experiments address the potential molecular mechanisms underlying these regeneration/repair and cellular defects, and data will be presented.
LB1000
LB1001
Differentiation of dermal papilla cells into a myogenic lineage for the treatment of duchenne muscular dystrophy M Rashidi1, N Rufaut2, L Jones3, T Partridge4 and R Sinclair5 1 The George Washington University, McLean, VA, 2 University of Melbourne, Dunedin, New Zealand, 3 University of Melbourne, Victoria, Australia, 4 The George Washington University, Washington, DC and 5 University of Melbourne, Melbourne, Victoria, Australia Duchenne muscular dystrophy (DMD) is the commonest muscular dystrophy caused by the absence of dystrophin. Stem cell therapy in DMD is one of the promising approaches for treatment. Multipotent stem cells residing in the hair follicle papilla are highly plastic. We showed that dermal papilla cells (DPC) undergo myogenic differentiation when co-cultured with different types of myoblasts including dystrophic human myoblasts. DPC incorporated into myotubes and upregulated the muscle marker, myogenin, in the co-culture with human myoblasts. DPC incorporation efficiency was low (< 5%) in all co-cultures and differed significantly (p value 0.001) between various types of myoblasts; however, no significant difference was observed between normal and dystrophic human myoblasts (p value 0.001). These encouraging findings suggested that the altered properties of dystrophic human myoblasts did not compromise the myogenic differentiation of DPC in vitro, supporting their in vivo application and possible therapeutic potential. The in vitro effects of galectin-1 and activation of Shh signaling pathway via recombinant Shh (rShh) and purmorphamine, on the myogenic differentiation of DPC, was also evaluated. None of the treatments increased myogenin expression in DPC; but, triggering Shh signaling produced a dose dependent pattern whereby the lower levels of signaling promoted myogenic differentiation while the higher levels inhibited it. Activating Shh signaling upstream of Smo, via purmorphamine, induced a biphasic differentiative response; however, the application of rShh hindered the differentiation of both cell types. Thus, murine DPC are a readily accessible source of stem cells that can undergo myogenic differentiation in vitro and their myogenic differentiation can be enhanced with proper treatment .
B12 Journal of Investigative Dermatology (2017), Volume 137
In vitro examination of Mexican hair shaft morphology using optical coherence tomography M Perper1, M Martinez2, A Maddy1, J Cervantes1, A Eber1, S Verne1, N Vazquez3, K Nouri1 and A Tosti1 1 University of Miami Miller School of Medicine, Miami, FL, 2 University of Miami Miller School of Medicine, Mexico City, Mexico and 3 University of Miami Miller School of Medicine, Monterrey, Mexico Hair diameter, shape, mechanical properties, combability, and moisture content have been previously examined, however primarily in Caucasian, African, and Asian individuals. We describe the distinct cross-sectional and morphological hair characteristics of Hispanics, an ethnic group accounting for 16% of total hair product sales in the United States, yet one that is not sufficiently studied. The VivoSightÒ swept-source multi-beam optical coherence tomography (OCT) system was used to evaluate hair diameter and shape in 30 female volunteers aged 18 to 55 recruited from Hospital Me´dica Sur in Mexico. Three hair samples from each volunteer were measured transversely along three distances, generating 9 crosssectional images, 2 measurements per image, and 18 measurements per patient for a total of 540 diameter measurements in 30 patients. Samples were measured 10 mm from the proximal portion of the fiber, middle position (equidistant point between the root and end), and 10 mm from the distal portion of the fiber. The minimum hair diameter of all measurements (n ¼ 540) was 0.06 mm while the maximum was 0.14 mm. The mean diameter of the hair samples was 0.10 0.01 mm. Of note, we found that Mexican hair resembles Caucasian and Asian hair, in that hair shafts tend to be round-shaped with homogenous diameters throughout the fiber, rather than elliptical, flattened, and irregular, as seen with Afro-ethnic hair. Mexican hair is similar to Asian hair in terms of diameter and shape, and thicker on average than Caucasian hair. Our findings suggest that OCT may be a viable method of hair examination in addition to more traditional approaches such as light, confocal, scanning electron, and transmission electron microscopy. Retailers may use information garnered from this study to tailor hair products to the unique hair characteristics of Hispanics, an ethnic group driving sales in the hair care market.