RESUMO
PURPOSE: In postoperative wound healing after surgical operations or ablative laser treatments, recent studies suggest the timely use of non-ablative fractional laser treatments with the aim to improve wound healing and prevent pathological scar formation. However, the underlying molecular mechanisms are poorly understood. The aim of this study was to investigate the effects of laser-assisted scar healing (LASH) at the molecular level and to combine it with already established wound healing-promoting local treatments. METHODS: We irradiated full-thickness 3D skin models with a fractional ablative Er:YAG laser to set standardized lesions to the epidermal and upper dermal layer. Subsequently, LASH was induced by irradiating the models with either a fractional non-ablative 1540 nm Er:Glass or 1550 nm diode laser. In addition, we tested the combination of non-ablative fractional laser treatment and topical aftercare with a dexpanthenol-containing ointment (DCO). RESULTS: Histological analysis revealed that models irradiated with the 1540 nm Er:Glass or 1550 nm diode laser exhibited accelerated but not complete wound closure after 16 h. In contrast, additional topical posttreatment with DCO resulted in complete wound closure. At gene expression level, both non-ablative laser systems showed similar effects on epidermal differentiation and mild anti-inflammatory properties. The additional posttreatment with DCO enhanced the wound-healing effects of LASH, especially the upregulation of epidermal differentiation markers and anti-inflammatory cytokines at the gene expression level. CONCLUSION: This in vitro study deciphers the biological effects of LASH with a fractional non-ablative 1540 nm Er:Glass or a 1550 nm diode laser in 3D skin models. These data help to better understand the biological properties of the LASH technique and is important to optimize its application.
Assuntos
Terapia a Laser , Lasers de Estado Sólido , Humanos , Cicatriz/metabolismo , Lasers Semicondutores/uso terapêutico , Pele/metabolismo , Cicatrização , Lasers de Estado Sólido/uso terapêutico , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Terapia a Laser/métodosRESUMO
Polychlorinated biphenyls (PCBs) are well known carcinogenic persistent environmental pollutants and endocrine disruptors. Our aim was to identify the possible dysregulation of genes in PCB exposed peripheral blood mononuclear cells (PBMCs) in order to give more insight into the differential pathophysiological effects of PCB congeners and mixtures, with an emphasis on immunological effects and oxidative stress. The PBMCs of a healthy volunteer (male, 56 years old) were exposed to a mixture of dioxin-like (DL)-PCBs (PCB 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, and 189, 250 µg/L resp.) or non-dioxin-like (NDL)-PCBs (PCB 28, 52, 101, 138, 153, 180, 250 µg/L resp.) or single PCB congener (no.28, 138, 153, 180, 250 µg/L resp.). After an incubation period of 24 h, a microarray gene expression screening was performed, and the results were compared to gene expression in control samples (PBMCs treated with the vehicle iso-octane). Treatment of PBMCs with the DL-PCB mixture resulted in the largest number of differentially regulated genes (181 upregulated genes >2-fold, 173 downregulated >2-fold). Treatment with the NDL-PCB mix resulted in 32 upregulated genes >2-fold and 12 downregulated genes >2-fold. A gene set enrichment analysis (GSEA) on DL-PCB treated PBMCs resulted in an upregulation of 125 gene sets and a downregulation of 76 gene sets. Predominantly downregulated gene sets were involved in immunological pathways (such as response to virus, innate immune response, defense response). An upregulation of pathways related to oxidative stress could be observed for all PCB congeners except PCB-28; the latter congener dysregulated the least number of genes. Our experiment augments the information known about immunological and cellular stress responses following DL- as well as NDL-PCB exposure and provides new information on PCB 28. Further studies should be performed to evaluate how disruption of these pathways contributes to the development of autoimmune diseases and cancer.
Assuntos
Carcinógenos/toxicidade , Dioxinas/toxicidade , Disruptores Endócrinos/toxicidade , Poluentes Ambientais/toxicidade , Expressão Gênica/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Células Cultivadas , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Immunosuppression is often considered as an indication for antibiotic prophylaxis to prevent surgical site infections (SSI) while performing skin surgery. However, the data on the risk of developing SSI after dermatologic surgery in immunosuppressed patients are limited. PATIENTS AND METHODS: All patients of the Department of Dermatology and Allergology at the University Hospital of RWTH Aachen in Aachen, Germany, who underwent hospitalization for a dermatologic surgery between June 2016 and January 2017 (6 months), were followed up after surgery until completion of the wound healing process. The follow-up addressed the occurrence of SSI and the need for systemic antibiotics after the operative procedure. Immunocompromised patients were compared with immunocompetent patients. The investigation was conducted as a retrospective analysis of patient records. RESULTS: The authors performed 284 dermatologic surgeries in 177 patients. Nineteen percent (54/284) of the skin surgery was performed on immunocompromised patients. The most common indications for surgical treatment were nonmelanoma skin cancer and malignant melanomas. Surgical site infections occurred in 6.7% (19/284) of the cases. In 95% (18/19), systemic antibiotic treatment was needed. Twenty-one percent of all SSI (4/19) were seen in immunosuppressed patients. CONCLUSION: According to the authors' data, immunosuppression does not represent a significant risk factor for SSI after dermatologic surgery. However, larger prospective studies are needed to make specific recommendations on the use of antibiotic prophylaxis while performing skin surgery in these patients.
Assuntos
Procedimentos Cirúrgicos Dermatológicos/efeitos adversos , Hospedeiro Imunocomprometido , Terapia de Imunossupressão/efeitos adversos , Neoplasias Cutâneas/cirurgia , Infecção da Ferida Cirúrgica/etiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Ceratose Actínica/cirurgia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Infecção da Ferida Cirúrgica/tratamento farmacológico , Adulto JovemRESUMO
Polychlorinated biphenyls (PCBs) are well- known man-made persistent environmental pollutants and endocrine disruptors. As a result of mass production in the past, background levels of these compounds can be measured in human blood worldwide. In 2010 high internal levels of PCBs were discovered in workers of a transformer-recycling company in Germany. Our aim was to measure, whether the expression of CYP1A1, CYP1B1, and IL-1ß is dysregulated in peripheral blood mononuclear cells (PBMCs) of the exposed individuals (nâ¯=â¯max 308). Further, we measured the regulation of CYP1A1, CYP1B1, AHRR (aromatic hydrocarbon receptor repressor) and IL-1ß in skin samples of 25 workers with elevated plasma PCB levels using quantitative PCR (q-RT-PCR). We found a significant correlation between the regulation of IL-1ß in skin samples and lipid adjusted PCB levels. In the PBMCs, the expression levels of CYP1A1, CYP1B1 and IL-1ß decreased over time with decreasing PCB plasma levels. The upregulation of the cytokine IL-1ß in exposed individuals with higher PCB plasma levels warrants further investigation in order to examine its role in the pathophysiology of autoimmune disorders and tumor promotion.
Assuntos
Biomarcadores/metabolismo , Exposição Ambiental/estatística & dados numéricos , Poluentes Ambientais/metabolismo , Bifenilos Policlorados/metabolismo , Pele/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Alemanha , Humanos , Leucócitos Mononucleares/metabolismo , Receptores de Hidrocarboneto ArílicoRESUMO
Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL-31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL-31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide-containing water-in-oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide-containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and ß4-integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide-containing skin care ointment reduced IL-31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.
Assuntos
Ceramidas/farmacologia , Fármacos Dermatológicos/farmacologia , Interleucinas/farmacologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Perda Insensível de Água/efeitos dos fármacos , Órgãos Bioartificiais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fibroblastos/metabolismo , Proteínas Filagrinas , Humanos , Queratinócitos/metabolismo , Bases para Pomadas , Pomadas , Proteínas Recombinantes/farmacologia , Água/metabolismoRESUMO
Polychlorinated biphenyls (PCB) are well known persistent and toxic environmental pollutants. Our aim was to identify effects of moderate-high exposure to dioxin-like (dl) and non-dioxin-like (ndl)-PCBs on the skin in order to provide more insight in the pathophysiological effects of these compounds. We performed a dermatological examination on 92 former workers from a transformer recycling company with known elevated serum PCB and/or dioxin (polychlorinated dibenzo-p-dioxin/polychlorinated dibenzo-p-furan (PCDD/F)) levels. In addition, we performed a skin cancer screening over a period of seven years (2010-2016) on resp. 268, 271, 210, 149, 92, 129 and 79 participants. We found a higher incidence of acne and malignancies of the skin (malignant melanoma, basal cell carcinoma and mycosis fungoides) in the workers compared to normal population. The probability of having hyperpigmentation on the skin was statistically significantly higher in workers with higher sumPCBs- (OR:1.09(1.12-2.17)), dioxin-like (dl)-PCBs- (OR:1.56(1.12-2.17)) and dioxin (PCDD/Fs) (OR:1.09(1.02-1.16)) levels. Age was a confounding factor in this model. Formation of hyperpigmentation could be an indicator for (moderate-high) exposure to toxic compounds like PCBs. The higher incidence of cutaneous malignancies found in the workers might be associated with PCB- and dioxin exposure, warranting further investigation on larger cohorts.
Assuntos
Benzofuranos , Dioxinas , Poluentes Ambientais , Hiperpigmentação , Bifenilos Policlorados , Dibenzodioxinas Policloradas , Neoplasias Cutâneas , Adulto , Idoso , Benzofuranos/toxicidade , Dibenzofuranos Policlorados/toxicidade , Poluentes Ambientais/toxicidade , Feminino , Humanos , Hiperpigmentação/epidemiologia , Incidência , Masculino , Pessoa de Meia-Idade , Bifenilos Policlorados/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Neoplasias Cutâneas/epidemiologiaRESUMO
The proinflammatory cytokine IL-36γ is highly expressed in epithelial cells and is a pivotal mediator of epithelial inflammation. In particular, IL-36γ is strongly associated with the inflammatory skin disease psoriasis. As with other IL-1 cytokines, IL-36γ is expressed as an inactive precursor and must be processed by specific proteases to become bioactive. Our aim therefore was to identify protease/s capable of IL-36γ activation and explore the importance of this activation in psoriasis. Using a keratinocyte-based activity assay in conjunction with small-molecule inhibitors and siRNA gene silencing, cathepsin S was identified as the major IL-36γ-activating protease expressed by epithelial cells. Interestingly, cathepsin S activity was strongly up-regulated in samples extracted from psoriasis patients relative to healthy controls. In addition, IL-36γ-Ser18, identified as the main product of cathepsin S-dependent IL-36γ cleavage, induced psoriasiform changes in human skin-equivalent models. Together, these data provide important mechanistic insights into the activation of IL-36γ and highlight that cathepsin S-mediated activation of IL-36γ may be important in the development of numerous IL-36γ-driven pathologies, in addition to psoriasis.
Assuntos
Catepsinas/metabolismo , Interleucina-1/genética , Psoríase/genética , Motivos de Aminoácidos , Catepsinas/genética , Linhagem Celular , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1/metabolismo , Psoríase/enzimologia , Psoríase/imunologiaRESUMO
BACKGROUND: Digital mucoid cysts have a tendency for recurrence after operative intervention. Several procedures are in use. OBJECTIVE: Retrospective evaluation for effectiveness, safety and patient satisfaction by using a questionnaire after treatment for digital mucoid cysts with targeted surgical excision and closure by flap-design. MATERIALS AND METHODS: All patients treated with surgical excision for digital mucoid cysts at the Dermatology Department of the Ludwigshafen City Hospital between 2007 and 2011 were evaluated using a specially designed questionnaire. RESULTS: We evaluated 31 patients. The patient group consisted of 65% women, the median age was 61 years. Seventy-eight percent of patients with nail involvement had a marked improvement or complete resolution of this complaint after surgery. A few complications (e.g., redness, pain or hematoma) were observed after treatment, but no patients required oral antibiotics. Patient evaluation of cosmetic outcome revealed high satisfaction with the procedure, nevertheless recurrence of the digital mucoid cysts was observed in 22.5% of all cases. CONCLUSION: Surgical excision in treatment of digital mucoid cysts was shown to be effective and safe. However, possible advantages and disadvantages of this treatment option should be discussed with the patients before a decision on the kind of therapy is reached.
Assuntos
Cisto Sinovial/cirurgia , Adolescente , Adulto , Idoso , Procedimentos Cirúrgicos Dermatológicos/efeitos adversos , Procedimentos Cirúrgicos Dermatológicos/métodos , Feminino , Dedos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
The response of the skin to harmful environmental agents is shaped decisively by the status of the immune system. Keratinocytes constitutively express and secrete the chemokine-like mediator, macrophage migration inhibitory factor (MIF), more strongly than dermal fibroblasts, thereby creating a MIF gradient in skin. By using global and epidermis-restricted Mif-knockout (Mif-/- and K14-Cre+/tg; Miffl/fl) mice, we found that MIF both recruits and maintains antigen-presenting cells in the dermis/epidermis. The reduced presence of antigen-presenting cells in the absence of MIF was associated with accelerated and increased formation of nonmelanoma skin tumors during chemical carcinogenesis. Our results demonstrate that MIF is essential for maintaining innate immunity in skin. Loss of keratinocyte-derived MIF leads to a loss of control of epithelial skin tumor formation in chemical skin carcinogenesis, which highlights an unexpected tumor-suppressive activity of MIF in murine skin.-Brocks, T., Fedorchenko, O., Schliermann, N., Stein, A., Moll, U. M., Seegobin, S., Dewor, M., Hallek, M., Marquardt, Y., Fietkau, K., Heise, R., Huth, S., Pfister, H., Bernhagen, J., Bucala, R., Baron, J. M., Fingerle-Rowson, G. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.
Assuntos
Fatores Inibidores da Migração de Macrófagos/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Pele/citologia , Pele/imunologia , Animais , Antracenos/toxicidade , Antígenos CD/genética , Antígenos CD/metabolismo , Carcinogênese , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Queratinócitos/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Piperidinas/toxicidade , Piridinas/toxicidade , Receptores CXCR/genética , Receptores CXCR/metabolismoRESUMO
Tasisulam is a promising antitumor agent with complex pharmacology, which is used as an antiproliferative agent in patients with metastatic melanoma and other solid tumors. Phase 2 melanoma studies showed promising results but had to be stopped because of insufficient tasisulam clearance leading to toxic side effects. To reduce the negative effects of tasisulam, we synthesized a novel sulfonimidamide-based analog to evaluate its antiproliferative effects in comparison to the original compound by performing a cell proliferation assay in melanoma cell lines SKMel23 and A375. The results revealed that the analog had inhibitory effects on the proliferation comparable to tasisulam in both investigated cell lines. These results could contribute to a reduced toxicity of tasisulam and lead to further clinical trials in metastatic melanoma.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , MelanomaAssuntos
Ácido Aminolevulínico/análogos & derivados , Ceratose Actínica/tratamento farmacológico , Lasers de Gás/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Ácido Aminolevulínico/uso terapêutico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Terapia a Laser/métodos , FotoquimioterapiaRESUMO
INTRODUCTION: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling. RESULTS: We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment. CONCLUSION: We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in 'silent' metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Interferon-alfa/fisiologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Animais , Apresentação de Antígeno , Humanos , Imunoterapia , Interferon-alfa/farmacologia , Janus Quinases , Leucócitos Mononucleares , Masculino , Melanoma/tratamento farmacológico , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transplante de Neoplasias , Fosforilação , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Regulação para CimaRESUMO
Clinical experiences with non-ablative fractional erbium glass laser therapy have demonstrated promising results for dermal remodelling and for the indications of striae, surgical scars and acne scars. So far, molecular effects on human skin following treatment with these laser systems have not been elucidated. Our aim was to investigate laser-induced effects on skin morphology and to analyse molecular effects on gene regulation. Therefore, human three-dimensional (3D) organotypic skin models were irradiated with non-ablative fractional erbium glass laser systems enabling qRT-PCR, microarray and histological studies at same and different time points. A decreased mRNA expression of matrix metalloproteinases (MMPs) 3 and 9 was observed 3 days after treatment. MMP3 also remained downregulated on protein level, whereas the expression of other MMPs like MMP9 was recovered or even upregulated 5 days after irradiation. Inflammatory gene regulatory responses measured by the expression of chemokine (C-X-C motif) ligands (CXCL1, 2, 5, 6) and interleukin expression (IL8) were predominantly reduced. Epidermal differentiation markers such as loricrin, filaggrin-1 and filaggrin-2 were upregulated by both tested laser optics, indicating a potential epidermal involvement. These effects were also shown on protein level in the immunofluorescence analysis. This novel standardised laser-treated human 3D skin model proves useful for monitoring time-dependent ex vivo effects of various laser systems on gene expression and human skin morphology. Our study reveals erbium glass laser-induced regulations of MMP and interleukin expression. We speculate that these alterations on gene expression level could play a role for dermal remodelling, anti-inflammatory effects and increased epidermal differentiation. Our finding may have implications for further understanding of the molecular mechanism of erbium glass laser-induced effects on human skin.
Assuntos
Cicatriz/radioterapia , Lasers de Estado Sólido/uso terapêutico , Pele/efeitos da radiação , Cicatriz/patologia , Proteínas Filagrinas , Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Terapia com Luz de Baixa Intensidade/métodos , Modelos Biológicos , Pele/patologia , Técnicas de Cultura de TecidosRESUMO
Primary dendritic cells and myeloid cell lines are used to assess the skin sensitization hazard in in vitro approaches. The aryl hydrocarbon receptor (AhR) modulates expression of CYP enzymes which play a significant role in the bioactivation of various xenobiotics. These studies revealed a strong constitutive expression of the AhR in primary human monocytes, monocyte-derived immature dendritic cells (iDC) and cord blood-derived Langerhans cells (LC). In contrast, mRNA and protein expression of AhR was hardly detectable in the cell lines THP-1 and MUTZ-3. U937 cells and MUTZ-3-derived dendritic (MUTZ-DC) or Langerhans cells (MUTZ-LC) showed about half the expression of AhR compared to iDC. Incubation of cells with the specific AhR-inducer benzo[a]anthracene resulted in an upregulation of CYP and IL-1ß mRNA expression in primary monocytes and iDC. CYP1A1 but not CYP1B1 and IL-1ß expression was increased by benzo[a]anthracene in these cell lines except for U937 cells. AhR-independent CYP genes were not regulated by benzo[a]anthracene. Constitutive mRNA expression of other non AhR-dependent CYP enzymes was higher in some of the cell lines compared to the corresponding primary cells. This study demonstrates significant differences in expression and regulation of phase I genes in cell lines currently used for in vitro skin sensitization hazard assessment compared to primary cells. Additional studies are required regarding the combination of cutaneous xenobiotic metabolizing enzymes and APC-sensitization for the development of valid in vitro models for skin sensitization assessment.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Citocromo P-450 CYP1A1/genética , Regulação da Expressão Gênica , Células Mieloides , Receptores de Hidrocarboneto Arílico/metabolismo , Linhagem Celular , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Células Dendríticas , Dermatite de Contato , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células de Langerhans , Monócitos , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
BACKGROUND AND OBJECTIVE: At present, there is no standardized in vitro human skin model for wound healing. Therefore, our aim was to establish and characterize an in vitro/ex vivo three-dimensional (3D) wound healing model, which we employed to analyze the effects of dexpanthenol on wound healing and gene regulation. MATERIALS AND METHODS: The novel human 3D skin wound healing model using scaffold and collagen 3D organotypic skin equivalents was irradiated with a non-sequential fractional ultrapulsed CO2 laser. These standardized injured full-thickness skin equivalents enable qRT-PCR, microarray, and histological studies analyzing the effect of topically or systemically applied compounds on skin wound healing. RESULTS: These human laser-irradiated skin models were found to be appropriate for in vitro wound healing analysis. Topical treatment of skin wounds with a 5% dexpanthenol water-in-oil emulsion or two different 5% dexpanthenol oil-in-water emulsions clearly enhanced wound closure compared to laser-irradiated untreated control models. To find out whether this positive effect is caused by the active substance dexpanthenol, laser-irradiated skin models were cultured in calciumpantothenate containing medium (20 µg/ml) compared to skin equivalents cultured without calciumpantothenate. 3D models cultured in calciumpantothenate revealed considerably faster wound closure compared to the control models. Quantitative RT-PCR studies showed enhanced mRNA expression of MMP3, IL1α, keratin-associated protein 4-12 (KRTAP4-12), and decreased expression of S100A7 in laser-irradiated skin models cultured in medium containing calciumpantothenate. CONCLUSION: This novel standardized human 3D skin wound healing model proves useful for topical pharmacological studies on wound healing and reveals new insights into molecular mechanisms of dexpanthenol-mediated effects on wound healing. In addition, these novel 3D model systems can be used to monitor ex vivo effects of various laser systems on gene expression and morphology of human skin.
Assuntos
Lasers de Gás/uso terapêutico , Modelos Biológicos , Ácido Pantotênico/análogos & derivados , Cicatrização/efeitos dos fármacos , Células Cultivadas , Procedimentos Cirúrgicos Dermatológicos/métodos , Humanos , Ácido Pantotênico/farmacologia , Cicatrização/genéticaRESUMO
BACKGROUND/AIM: Vitamin A (all- trans -retinol, ATRol) serves as a precursor for all- trans -retinoic acid (ATRA), a ligand for the retinoic acid receptor (RAR), representing a potent regulator for many physiological processes. While murine melanoma cells are highly sensitive to retinoid treatment, human melanoma cells have developed still unidentified mechanisms that mediate cellular retinoid resistance. One of the key retinoid metabolizing enzymes is lecithin retinol acyltransferase (LRAT), which catalyzes the transformation of ATRol into inactive retinyl esters. LRAT is highly expressed in human melanoma cells. The aim of this study was to identify the mechanisms in retinol metabolism that are responsible for cellular retinoid sensitivity in the murine melanoma cell line B16F10. METHODS: mRNA expression analysis, cell viability assessment and determination of intracellular retinoid levels using HPLC analysis of a generated LRAT-overexpressing B16F10 cell line compared to the control B16F10 cell line. RESULTS: We found that the murine retinoid-sensitive B16F10 cell line does not express the enzyme LRAT. LRAT overexpression decreased the antiproliferative effects of retinoid treatment in these melanoma cells. The RAR-regulated enzyme Cyp26a1 showed a significantly lower expression in LRAT-overexpressing B16F10 cells. Cyp26a1 expression was restored after ATRA incubation. HPLC analysis revealed that the level of inactive retinyl ester increased after ATRol treatment, and levels of the substrate ATRol and biologically active ATRA significantly decreased in LRAT-overexpressing murine melanoma. Consistently with this, levels of 4-oxoretinoic acid, an ATRA metabolite and Cyp26a1 product, were also decreased in LRAT-overexpressing cells. CONCLUSION: Our results revealed a direct link between LRAT expression and regulation of ATRA levels indicating that the absence of LRAT-catalyzed retinol esterification is important for mediating retinoid sensitivity in murine melanoma cells. Thus, our data suggest that LRAT overexpression represents a novel mechanism by which tumor cells can escape high supplementary ATRA levels that mediate tumor-suppressive RAR signaling.
Assuntos
Aciltransferases/metabolismo , Melanoma Experimental/metabolismo , Retinaldeído/farmacologia , Tretinoína/farmacologia , Vitamina A/farmacologia , Aciltransferases/genética , Animais , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/genética , Camundongos , Retinaldeído/análogos & derivados , Ácido Retinoico 4 HidroxilaseRESUMO
Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.
Assuntos
Aciltransferases/genética , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Tretinoína/química , Catálise , Linhagem Celular Tumoral , Células Cultivadas , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos/citologia , Melanócitos/metabolismo , Vitamina A/química , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear. METHODS AND RESULTS: To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (-/-) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher. CONCLUSION: NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.
Assuntos
Apoptose/efeitos da radiação , Epiderme/metabolismo , Queratinócitos/metabolismo , Neuropilina-1/metabolismo , Raios Ultravioleta , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Células Epidérmicas , Epiderme/efeitos da radiação , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Chronic skin exposure to ultraviolet light stimulates the production of cytokines known to be involved in the initiation of skin cancer. Recent studies in mouse models suggested a role for macrophage migration inhibitory factor (MIF) in the UVB-induced pathogenesis of nonmelanoma skin cancer (NMSC). Our studies aimed at defining the pathophysiological function of MIF in cutaneous inflammatory reactions and in the development and progression of NMSC. Immunohistochemical analysis revealed a moderate expression of MIF in normal human skin samples but an enhanced expression of this cytokine in lesional skin of patients with actinic keratosis or cutaneous SCC. Enzyme-linked immunosorbent assay studies showed a time-dependent increase in MIF secretion after a moderate single-dose UVB irradiation in NHEKs and SCC tumor cells. MIF is known to interact with CXCR2, CXCR4 and CD74. These receptors are not constitutively expressed in keratinocytes and HaCaT cells and their expression is not induced by UVB irradiation either. However, stimulation with IFNγ upregulated CD74 surface expression in these cells. Affymetrix(®) Gene Chip analysis revealed that only keratinocytes prestimulated with IFNγ are responsive to MIF. These findings indicate that MIF may be an important factor in the pathogenesis of NMSC tumorigenesis and progression in an inflammatory environment.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Ceratose Actínica/metabolismo , Neoplasias Cutâneas/metabolismo , Pele/efeitos da radiação , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imuno-Histoquímica , Interferon gama/farmacologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Ceratose Actínica/genética , Ceratose Actínica/patologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Cultura Primária de Células , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Pele/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Raios UltravioletaRESUMO
Several transport proteins are constitutively expressed in skin cells, but the putative role of the ABC transporter P-glycoprotein (P-gp) in human skin is yet unknown. Therefore, we analysed mRNA and protein expression and localization of P-gp in human skin. Using qRT-PCR, we demonstrated a strong MDR1 mRNA expression in whole skin specimens and dermis, whereas the expression of MDR1 in epidermis, epidermal keratinocytes or dermal fibroblasts was only weak. Immunohistochemistry confirmed mRNA data and revealed a marked expression of P-gp within sweat ducts, vessels, nerve sheaths and muscles of human skin and a moderate expression in basal epidermis. Our findings closely correlate with previous studies in murine skin supporting the role of P-gp in the uptake of compounds from the epidermal compartment and their secretion into the bloodstream and sweat ducts. It may also prevent the uptake of xenobiotics into the skin by functioning as a barrier located in the dermal vasculature.