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LB820 Identification of a novel marker for cellular senescence
ABSTRACTS | LB816
LB817
Transdermal delivery of insulin using novel chemical penetration enhancers designed via in silico, non-linear QSPR modeling, utilizing genetic algorithms and artificial neural networks A Grada1, S Madihally2 and KA Gasem2 1 Dermatology, Boston University School of Medicine, Boston, MA and 2 Chemical Engineering, Oklahoma State University, Stillwater, OK Transdermal drug delivery (TDD) is gaining significance in the recent years due to its advantages over oral delivery and hypodermic needle injections. An insulin patch placed on the skin may be able to deliver sustained physiological levels of basal insulin; however, because insulin is a relatively large molecule, it does not penetrate the skin barrier easily. Using chemicals penetration enhancers (CPEs) is one of the promising ways to increase skin permeation. Previously, traditional chemical enhancers have had limited success in delivery of macromolecules and peptides such as insulin and vaccines. To resolve this challenge, we have reported on an effective methodology of predicting useful CPEs using genetic algorithms and neural networks for non-linear, quantitative structure-property relationship (QSPR) models based upon representative molecular properties. The predicted molecules were extensively tested for cytotoxicity and ex vivo transdermal delivery. The main objective of this study is to evaluate the in vivo efficacy of delivering insulin across the epidermal barrier with and without the newly designed CPEs in a streptozotocin (STZ)-induced diabetic rat model. These animals were randomly assigned to one of the following groups: (1) control (no CPE or insulin); (2) insulin alone (no CPE); (3) test CPE; (4) test CPE and insulin (at least n¼3 per group per CPE). Eight hours prior to application of the patch, all diabetic rats were transferred to individual metabolic cages and were fasted to normalize the glucose level. After application of the patch, blood glucose levels were monitored at different time points. After five days, we evaluated the overall body weight change, skin toxicity, and hepatotoxicity. Our findings through animal trials indicate proven safety and efficacy of the newly designed CPEs.
Immunomodulatory effects of nanoparticles in a mouse model of skin allergy S Jatana1, BC Palmer2, S Phelan2 and L DeLouise3,1,2 1 Biomedical Engineering, University of Rochester, Rochester, NY, 2 Toxicology, University of Rochester, Rochester, NY and 3 Dermatology, University of Rochester, Rochester, NY The cosmeceutical market is fast growing and valued to be at $31.84 billion in 2016. Many products designed to treat hair damage, hyperpigmentation, photoaging and wrinkles contain nanoparticles (NPs) such as liposomes, nanocapsules, nanocrystals, dendrimers, nanogold, nanosilver, cubosomes, niosomes and fullerenes. NPs are also incorporated into drug and vaccine delivery systems. Studies report that skin penetration depends on the NP physiochemical properties (size, shape, surface charge and composition) and the skin barrier status. Understanding the factors that affect NP skin penetration and their interaction with the cellular components in skin are critical for the tailoring and design of NP-based cosmetic and transdermal therapeutics. In this study we investigate the development of a topical therapeutic containing NPs using a mouse model of Allergic Contact Dermatitis (ACD). We discovered that some small (<100 nm) and negative charged NPs including gold NPs (AuNP), silver NPs (AgNP) and silica NPs (SiNP) have an intrinsic ability to suppress the inflammatory response in the elicitation phase of the allergic response to the common chemical sensitizer, dinitrofluorobenzene (DNFB). These NPs when applied within a 2 hour window of exposure to a DNFB reduced the influx of immune cells (neutrophils and T cells) and mitigate mast cell degranulation and the tissue cytokine milieu thereby generating an immunosuppressive effect. Importantly, we also observed a reduced swelling response when SiNPs (20 nm) were co-administered along with 2-deoxyurushiol, a skin irritant that resembles the chemical sensitizer in poison ivy. These findings suggest an opportunity to develop a NP based therapeutic for treating/preventing skin allergies. Future studies seek to examine the extensibility of our findings to other Th1 and Th2 sensitizing agents like 2-deoxyurushiol and to develop a cellular and molecular level understanding of the immunosuppressive mechanism.
LB818
LB819
Cosmetic sunscreen formulations provide protection against UVR-induced mitochondrial DNA damage in human skin cells A Bowman1, PJ Matts2, F Neuser3 and MA Birch-Machin1 1 Department of Dermatology, Newcastle University, Newcastle, United Kingdom, 2 Procter & Gamble, Egham, United Kingdom and 3 Procter & Gamble, Cincinnati, OH Mitochondrial DNA damage has been shown to be a reliable and sensitive biomarker of UVR exposure in human skin. In this study, the protective effect of a cosmetic sunscreen formulation with SPF30 and broadband UVA/UVB protection was analyzed using primary human skin cells (keratinocytes and fibroblasts), following ultraviolet irradiation at a range similar to that of sunlight. This UV range was obtained using 2 lamp types; (a) a solar simulator (Newport) and (b) Arimed B lamps in a sunbed. Mitochondrial DNA (mtDNA) was used as biomarker of solar simulated UV-induced damage in the skin cells. A control product without UV filters was also included in the study. The efficiency of these formulations to prevent UVinduced damage was analyzed by applying the products (2 mg/cm2) to Transpore tape covering Petri dishes containing primary human skin cells, followed by UV irradiation. With both fibroblasts and keratinocytes, mtDNA damage was observed when cells were irradiated with the Arimed B lamps in the presence of non-SPF formulation (P<0.0001) to a similar extent to that of tape alone (P<0.0001). Compared to foil-covered cells this was an approximately 2-fold difference in damage. In comparison, both cell types irradiated in the presence of the SPF30 formulation showed no increase in mtDNA damage as compared to the foil-covered cells (P>0.05). This suggests that the cosmetic sunscreen formulation with SPF30 cream is providing mtDNA protection to a similar extent as that provided by the foil at a dose of 2 SED. Similar results were obtained when cells were irradiated with the solar simulator; however mtDNA damage was induced to a lesser extent, possibly due to differences in exposure times between the 2 lamps. These results suggest that the tested SPF30 formulation is efficient at protecting human skin cells from sunlight-induced damage; future work would be required to determine whether this is a phenomenon also observed in human skin in vivo.
PD-1 inhibitor Nivolumab-induced squamous cell carcinoma A Jacobsen and J Strasswimmer Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL PD-1 inhibitors, approved for use in metastatic melanoma, have recently been reported as successful treatments for metastatic cutaneous squamous cell carcinoma (Lipson 2016, Chang 2015). The PD-1 inhibitors, pembrolizumab and nivolumab, can induce phototoxic eruptions, but until this report, have not been shown to promote SCC development. We now report a patient who received nivolumab for metastatic melanoma who subsequently developed cutaneous SCCs. A light skinned man in his seventies with metastatic melanoma received one course of nivolumab that was complicated by fevers, swelling, arthralgias, myalgias and a grade 3 phototoxic reaction. On exam, he had an exfoliative dermatitis on his hands, feet, and legs and also a photodistributed examthematous eruption on his arms and the V-neck of his chest, with sparing of the area where he normally wears a wristwatch. Significantly, he had been exclusively wearing high-quality long-sleeved UPF 50+ clothing, as defined by the American Society for Testing and Materials. He then developed six biopsyproven SCCs. Significantly, he had only one other SCC in the previous four years. Squamous cell carcinomas, keratoacanthomas and benign keratotic squamoproliferative lesions are known cutaneous adverse effects of other agents including the BRAF inhibitors vemurafenib and dabrafenib (MacDonald 2015), and the unique molecular pathogenesis has been described (Su 2012). However, PD-1 inhibitors have not previously been reported to cause these or other secondary malignancies. In our patient, the short latency of onset (<8 weeks) is similar to that seen with SCCs associated with BRAF inhibitors (Lacouture 2013), but the mechanism is likely to be different. This new adverse event may be important to take into account if PD-1 inhibitors become used more often for metastatic squamous cell carcinoma.
LB820
LB821
Identification of a novel marker for cellular senescence A Wang1, P Ong1, C Ho1, LJ Hor1, C Clavel3,2 and O Dreesen1,2 1 Cell Ageing, Institute of Medical Biology, Singapore, Singapore, 2 Nanyang Technological University, Lee Kong Chian School of Medicine, Singapore, Singapore and 3 Hair Pigmentation, Institute of Medical Biology, Singapore, Singapore Cellular senescence is an irreversible growth arrest that plays a critical role in regulating cellular lifespan in vitro and in vivo. Senescent cells accumulate with age in many tissues, including skin, in pre-neoplastic lesions and at sites of age-related pathologies. As such they limit tissue regeneration and contribute to various pathologies associated with ageing. Senescent cells can be identified by several criteria including loss of proliferative capacity, morphological changes, the appearance of senescence-associated heterochromatin foci, activation of DNA damage response pathways, secretion of inflammatory factors and the presence of histologically-detectable senescence-associated b-galactosidase (SA-b-gal) activity. However, most the majority of senescence markers are not specific. In addition, it remains unclear how these markers mechanistically contribute to the senescence phenotype. Our goal was to identify novel biomarkers to identify different senescent cell types. To this end, we used cells from patients with progeria, an accelerated ageing syndrome, to identify novel senescence markers. We found lamin B1 reduction as a hallmark of senescent human keratinocytes, fibroblasts and melanocytes, as well as during chronological ageing of human skin. In addition, lamin B1 levels decreased upon induction of oncogene-induced senescence (by doxycycline-inducible expression of BRAFV600E). To investigate whether this marker could be used to quantify the impact of UV exposure on senescence, we exposed human cell types to various doses of UV and quantified their exposure by measuring lamin B1 protein levels. Our results demonstrate that lamin B1 is a useful marker to identify senescent cells in vitro and in vivo e and to quantify the impact of environmental factors including UV on cellular ageing.
B12 Journal of Investigative Dermatology (2016), Volume 136
Critical role of Wnt signaling in follicular melanocyte stem cells Q Sun1, H Hu1, M Takeo1, W Lee1, MM Taketo2, SE Millar3 and M Ito1 1 Dermatology, New York University, New York, NY, 2 Kyoto University, Kyoto, Japan and 3 University of Pennsylvania, Philadelphia, PA Wnt signal controls stem cell behavior during homeostasis, regeneration and cancer in a variety of tissues in the body. How this signaling pathway impacts stem cells in the melanocytic lineage is not completely understood. Our study reveals that extrinsic Wnt ligands produced by epithelial niche cells are required for the function of melanocyte stem cells (McSCs) that are responsible for hair pigmentation. Upon exposure to Wnt ligands at the initiation of hair regeneration, McSCs give rise to mature melanocytes that produce pigment for the hair. Loss and gain of function of Wnt signaling in McSCs revealed that temporal activation of Wnt signaling promotes McSC differentiation and simultaneously maintains their self-renewing capacity. We also found that McSCs undergo prolonged Wnt activation during aging. This causes the loss of their self-renewing capacity similar to what is seen with constitutive Wnt activation in McSCs. In contrast, once McSCs undergo malignant transformation induced by oncogenic mutations, constitutive Wnt activation promotes their differentiation without diminishing the self-renewing capacity. This results in an aggressive melanoma with a highly differentiated melanocytic phenotype. These results show that the Wnt signaling pathway is critical to fine-tune the balance between self-renewal and differentiation of McSCs, and these regulatory mechanisms are dysregulated during cancerous transformation of McSCs.
LB817
Transdermal delivery of insulin using novel chemical penetration enhancers designed via in silico, non-linear QSPR modeling, utilizing genetic algorithms and artificial neural networks A Grada1, S Madihally2 and KA Gasem2 1 Dermatology, Boston University School of Medicine, Boston, MA and 2 Chemical Engineering, Oklahoma State University, Stillwater, OK Transdermal drug delivery (TDD) is gaining significance in the recent years due to its advantages over oral delivery and hypodermic needle injections. An insulin patch placed on the skin may be able to deliver sustained physiological levels of basal insulin; however, because insulin is a relatively large molecule, it does not penetrate the skin barrier easily. Using chemicals penetration enhancers (CPEs) is one of the promising ways to increase skin permeation. Previously, traditional chemical enhancers have had limited success in delivery of macromolecules and peptides such as insulin and vaccines. To resolve this challenge, we have reported on an effective methodology of predicting useful CPEs using genetic algorithms and neural networks for non-linear, quantitative structure-property relationship (QSPR) models based upon representative molecular properties. The predicted molecules were extensively tested for cytotoxicity and ex vivo transdermal delivery. The main objective of this study is to evaluate the in vivo efficacy of delivering insulin across the epidermal barrier with and without the newly designed CPEs in a streptozotocin (STZ)-induced diabetic rat model. These animals were randomly assigned to one of the following groups: (1) control (no CPE or insulin); (2) insulin alone (no CPE); (3) test CPE; (4) test CPE and insulin (at least n¼3 per group per CPE). Eight hours prior to application of the patch, all diabetic rats were transferred to individual metabolic cages and were fasted to normalize the glucose level. After application of the patch, blood glucose levels were monitored at different time points. After five days, we evaluated the overall body weight change, skin toxicity, and hepatotoxicity. Our findings through animal trials indicate proven safety and efficacy of the newly designed CPEs.
Immunomodulatory effects of nanoparticles in a mouse model of skin allergy S Jatana1, BC Palmer2, S Phelan2 and L DeLouise3,1,2 1 Biomedical Engineering, University of Rochester, Rochester, NY, 2 Toxicology, University of Rochester, Rochester, NY and 3 Dermatology, University of Rochester, Rochester, NY The cosmeceutical market is fast growing and valued to be at $31.84 billion in 2016. Many products designed to treat hair damage, hyperpigmentation, photoaging and wrinkles contain nanoparticles (NPs) such as liposomes, nanocapsules, nanocrystals, dendrimers, nanogold, nanosilver, cubosomes, niosomes and fullerenes. NPs are also incorporated into drug and vaccine delivery systems. Studies report that skin penetration depends on the NP physiochemical properties (size, shape, surface charge and composition) and the skin barrier status. Understanding the factors that affect NP skin penetration and their interaction with the cellular components in skin are critical for the tailoring and design of NP-based cosmetic and transdermal therapeutics. In this study we investigate the development of a topical therapeutic containing NPs using a mouse model of Allergic Contact Dermatitis (ACD). We discovered that some small (<100 nm) and negative charged NPs including gold NPs (AuNP), silver NPs (AgNP) and silica NPs (SiNP) have an intrinsic ability to suppress the inflammatory response in the elicitation phase of the allergic response to the common chemical sensitizer, dinitrofluorobenzene (DNFB). These NPs when applied within a 2 hour window of exposure to a DNFB reduced the influx of immune cells (neutrophils and T cells) and mitigate mast cell degranulation and the tissue cytokine milieu thereby generating an immunosuppressive effect. Importantly, we also observed a reduced swelling response when SiNPs (20 nm) were co-administered along with 2-deoxyurushiol, a skin irritant that resembles the chemical sensitizer in poison ivy. These findings suggest an opportunity to develop a NP based therapeutic for treating/preventing skin allergies. Future studies seek to examine the extensibility of our findings to other Th1 and Th2 sensitizing agents like 2-deoxyurushiol and to develop a cellular and molecular level understanding of the immunosuppressive mechanism.
LB818
LB819
Cosmetic sunscreen formulations provide protection against UVR-induced mitochondrial DNA damage in human skin cells A Bowman1, PJ Matts2, F Neuser3 and MA Birch-Machin1 1 Department of Dermatology, Newcastle University, Newcastle, United Kingdom, 2 Procter & Gamble, Egham, United Kingdom and 3 Procter & Gamble, Cincinnati, OH Mitochondrial DNA damage has been shown to be a reliable and sensitive biomarker of UVR exposure in human skin. In this study, the protective effect of a cosmetic sunscreen formulation with SPF30 and broadband UVA/UVB protection was analyzed using primary human skin cells (keratinocytes and fibroblasts), following ultraviolet irradiation at a range similar to that of sunlight. This UV range was obtained using 2 lamp types; (a) a solar simulator (Newport) and (b) Arimed B lamps in a sunbed. Mitochondrial DNA (mtDNA) was used as biomarker of solar simulated UV-induced damage in the skin cells. A control product without UV filters was also included in the study. The efficiency of these formulations to prevent UVinduced damage was analyzed by applying the products (2 mg/cm2) to Transpore tape covering Petri dishes containing primary human skin cells, followed by UV irradiation. With both fibroblasts and keratinocytes, mtDNA damage was observed when cells were irradiated with the Arimed B lamps in the presence of non-SPF formulation (P<0.0001) to a similar extent to that of tape alone (P<0.0001). Compared to foil-covered cells this was an approximately 2-fold difference in damage. In comparison, both cell types irradiated in the presence of the SPF30 formulation showed no increase in mtDNA damage as compared to the foil-covered cells (P>0.05). This suggests that the cosmetic sunscreen formulation with SPF30 cream is providing mtDNA protection to a similar extent as that provided by the foil at a dose of 2 SED. Similar results were obtained when cells were irradiated with the solar simulator; however mtDNA damage was induced to a lesser extent, possibly due to differences in exposure times between the 2 lamps. These results suggest that the tested SPF30 formulation is efficient at protecting human skin cells from sunlight-induced damage; future work would be required to determine whether this is a phenomenon also observed in human skin in vivo.
PD-1 inhibitor Nivolumab-induced squamous cell carcinoma A Jacobsen and J Strasswimmer Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL PD-1 inhibitors, approved for use in metastatic melanoma, have recently been reported as successful treatments for metastatic cutaneous squamous cell carcinoma (Lipson 2016, Chang 2015). The PD-1 inhibitors, pembrolizumab and nivolumab, can induce phototoxic eruptions, but until this report, have not been shown to promote SCC development. We now report a patient who received nivolumab for metastatic melanoma who subsequently developed cutaneous SCCs. A light skinned man in his seventies with metastatic melanoma received one course of nivolumab that was complicated by fevers, swelling, arthralgias, myalgias and a grade 3 phototoxic reaction. On exam, he had an exfoliative dermatitis on his hands, feet, and legs and also a photodistributed examthematous eruption on his arms and the V-neck of his chest, with sparing of the area where he normally wears a wristwatch. Significantly, he had been exclusively wearing high-quality long-sleeved UPF 50+ clothing, as defined by the American Society for Testing and Materials. He then developed six biopsyproven SCCs. Significantly, he had only one other SCC in the previous four years. Squamous cell carcinomas, keratoacanthomas and benign keratotic squamoproliferative lesions are known cutaneous adverse effects of other agents including the BRAF inhibitors vemurafenib and dabrafenib (MacDonald 2015), and the unique molecular pathogenesis has been described (Su 2012). However, PD-1 inhibitors have not previously been reported to cause these or other secondary malignancies. In our patient, the short latency of onset (<8 weeks) is similar to that seen with SCCs associated with BRAF inhibitors (Lacouture 2013), but the mechanism is likely to be different. This new adverse event may be important to take into account if PD-1 inhibitors become used more often for metastatic squamous cell carcinoma.
LB820
LB821
Identification of a novel marker for cellular senescence A Wang1, P Ong1, C Ho1, LJ Hor1, C Clavel3,2 and O Dreesen1,2 1 Cell Ageing, Institute of Medical Biology, Singapore, Singapore, 2 Nanyang Technological University, Lee Kong Chian School of Medicine, Singapore, Singapore and 3 Hair Pigmentation, Institute of Medical Biology, Singapore, Singapore Cellular senescence is an irreversible growth arrest that plays a critical role in regulating cellular lifespan in vitro and in vivo. Senescent cells accumulate with age in many tissues, including skin, in pre-neoplastic lesions and at sites of age-related pathologies. As such they limit tissue regeneration and contribute to various pathologies associated with ageing. Senescent cells can be identified by several criteria including loss of proliferative capacity, morphological changes, the appearance of senescence-associated heterochromatin foci, activation of DNA damage response pathways, secretion of inflammatory factors and the presence of histologically-detectable senescence-associated b-galactosidase (SA-b-gal) activity. However, most the majority of senescence markers are not specific. In addition, it remains unclear how these markers mechanistically contribute to the senescence phenotype. Our goal was to identify novel biomarkers to identify different senescent cell types. To this end, we used cells from patients with progeria, an accelerated ageing syndrome, to identify novel senescence markers. We found lamin B1 reduction as a hallmark of senescent human keratinocytes, fibroblasts and melanocytes, as well as during chronological ageing of human skin. In addition, lamin B1 levels decreased upon induction of oncogene-induced senescence (by doxycycline-inducible expression of BRAFV600E). To investigate whether this marker could be used to quantify the impact of UV exposure on senescence, we exposed human cell types to various doses of UV and quantified their exposure by measuring lamin B1 protein levels. Our results demonstrate that lamin B1 is a useful marker to identify senescent cells in vitro and in vivo e and to quantify the impact of environmental factors including UV on cellular ageing.
B12 Journal of Investigative Dermatology (2016), Volume 136
Critical role of Wnt signaling in follicular melanocyte stem cells Q Sun1, H Hu1, M Takeo1, W Lee1, MM Taketo2, SE Millar3 and M Ito1 1 Dermatology, New York University, New York, NY, 2 Kyoto University, Kyoto, Japan and 3 University of Pennsylvania, Philadelphia, PA Wnt signal controls stem cell behavior during homeostasis, regeneration and cancer in a variety of tissues in the body. How this signaling pathway impacts stem cells in the melanocytic lineage is not completely understood. Our study reveals that extrinsic Wnt ligands produced by epithelial niche cells are required for the function of melanocyte stem cells (McSCs) that are responsible for hair pigmentation. Upon exposure to Wnt ligands at the initiation of hair regeneration, McSCs give rise to mature melanocytes that produce pigment for the hair. Loss and gain of function of Wnt signaling in McSCs revealed that temporal activation of Wnt signaling promotes McSC differentiation and simultaneously maintains their self-renewing capacity. We also found that McSCs undergo prolonged Wnt activation during aging. This causes the loss of their self-renewing capacity similar to what is seen with constitutive Wnt activation in McSCs. In contrast, once McSCs undergo malignant transformation induced by oncogenic mutations, constitutive Wnt activation promotes their differentiation without diminishing the self-renewing capacity. This results in an aggressive melanoma with a highly differentiated melanocytic phenotype. These results show that the Wnt signaling pathway is critical to fine-tune the balance between self-renewal and differentiation of McSCs, and these regulatory mechanisms are dysregulated during cancerous transformation of McSCs.