Here, we verify the depigmenting action of Pouteria macrophylla fruit extract (EXT), incorporate it into a safe topical microemulsion and assess its effectiveness in a 3D pigmented skin model. Melanocytes-B16F10- were used to assess the EXT effects on cell viability, melanin synthesis, and melanin synthesis-related gene transcription factor expression, which demonstrated a 32% and 50% reduction of intra and extracellular melanin content, respectively. The developed microemulsion was composed of Cremophor EL®/Span 80 4:1 (w/w), ethyl oleate, and pH 4.5 HEPES buffer and had an average droplet size of 40 nm (PdI 0.40 ± 0.07). Skin irritation test with reconstituted epidermis (Skin Ethic RHETM) showed that the formulation is non-irritating. Tyrosinase inhibition was maintained after skin permeation in vitro, in which microemulsion showed twice the inhibition of the conventional emulsion (20.7 ± 2.2% and 10.7 ± 2.4%, respectively). The depigmenting effect of the microemulsion was finally confirmed in a 3D culture model of pigmented skin, in which histological analysis showed a more pronounced effect than a commercial depigmenting formulation. In conclusion, the developed microemulsion is a promising safe formulation for the administration of cutite fruit extract, which showed remarkable depigmenting potential compared to a commercial formulation.

Patients infected with the Dengue virus (DENV) often present with a massive generation of DENV-specific antibody-secreting cells (ASCs) in the blood. In some cases, these ASCs represent more than 50% of the circulating B cells, a higher magnitude than those induced by other infections, vaccinations, and plasma cell lymphomas. However, it remains unclear how the DENV infection elicits this colossal response. To address this issue, we utilised an in vitro strategy to induce human PBMCs of healthy individuals incubated with DENV particles (DENV4 TVP/360) to differentiate into ASCs. As controls, PBMCs were incubated with a mitogen cocktail or supernatants of uninfected C6/36 cells (mock). The ASC phenotype and function were increasingly detected in the DENV and mitogen-cultured PBMCs as compared to mock-treated cells. In contrast to the in vivo condition, secreted IgG derived from the PBMC-DENV culture was not DENV-specific. Lower ASC numbers were observed when inactivated viral particles or purified B cells were added to the cultures. The physical contact was essential between B cells and the remaining PBMCs for the DENV-mediated ASC response. Considering the evidence for the activation of the tryptophan metabolism detected in the serum of Dengue patients, we assessed its relevance in the DENV-mediated ASC differentiation. For this, tryptophan and its respective metabolites were quantified in the supernatants of cell cultures through mass spectrophotometry. Tryptophan depletion and kynurenine accumulation were found in the supernatants of PBMC-DENV cultures, which presented enhanced detection of indoleamine 2,3-dioxygenase 1 and 2 transcripts as compared to controls. In PBMC-DENV cultures, tryptophan and kynurenine levels strongly correlated to the respective ASC numbers, while the kynurenine levels were directly proportional to the secreted IgG titers. Contrastingly, PBMCs incubated with Zika or attenuated Bonezi et al. Dengue-Mediated Antibody-Secreting Cell DifferentiationYellow Fever viruses showed no correlation between their kynurenine concentrations and ASC numbers. Therefore, our data revealed the existence of distinct pathways for the DENV-mediated ASC differentiation and suggest the involvement of the tryptophan metabolism in this cellular process triggered by flavivirus infections.

Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co‐cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN‐γ. Since increased KYN production depends on the IFN‐γ‐inducible enzyme indoleamine‐2,3‐dioxygenase (IDO), we propose that part of the effect of IFN‐γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH4Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH4Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation.

Efeitos dos inibidores de IDO e TDO na proliferação, migração e invasão de melanomas humanos e na atividade tumoricida de células mononucleares. Maryana Stephany Ferreira Branquinho Versão corrigida da Dissertação conforme resolução CoPGr 6018. O original encontra-se disponível no Serviço de Pós Graduação da FCF/USP. Dissertação para obtenção do Título de Mestre Orientador: Prof. Tit. Ana Campa São Paulo 2015 1 . Bioquímica clínica : Medicina 2. Câncer : Imunologia I. T . II. Campa, Ana, orientador. 616.0756 CDD Aos meus pais, Sivaldo e Suelene. Obrigada por acreditarem e incentivarem esta conquista. À Ana Campa, orientadora, e definição melhor não há. Obrigada por abrir os meus olhos para a ciência. "É preciso força pra sonhar e perceber que a estrada vai além do que se vê..." ♪ Agradecimentos Primeiramente agradeço a Deus! Obrigada por tornar possível esta vitória! "Deus nos concede, a cada dia, uma página de vida no livro do tempo. Aquilo que colocamos nela corre por nossa conta". Chico Xavier Mãe e Pai: Obrigada! Não existem palavras que eu invente para escrever aqui capazes de expressar a minha gratidão. Talvez a maneira na qual eu irei olhar pra vocês no final da minha aula no dia da defesa, traduza este sentimento. Nada disso aconteceria se vocês não tivessem dito "ok, pode deixar tudo aqui com a gente, pode ir para SP.". Obrigada por serem os melhores pais e os melhores amigos. Vocês são os melhores do mundo! Guilherme, obrigada por sempre ter sido o meu maior parça e o melhor irmão que alguém pode ter! Espero que você me ajude a fazer na comemoração da defesa, metade do que fizemos na da qualificação. Só metade porque não há necessidade de apelação dessa vez, hehehe. Madrinha, obrigada por estar sempre aqui com a gente, sem a senhora a vida não teria o mesmo sentido. E obrigada também pela oncinha que eu ganhava a cada ida à Alfenas, ela salvava todo final de mês (e vai continuar salvando no doutorado). Vocês quatro são a melhor família para a qual Deus poderia ter me mandado. EU AMO VCS! Chefa, obrigada pela paciência, dedicação e confiança durante esses três anos. Ahhh, e obrigada também pelo abraço com o qual você me recebeu quando vim conhecer o Lab. Ele fez toda a diferença no momento de resolver se era isso mesmo que eu queria. Ao pessoal do Lab: Ale, Ari, Carol, Edson, Fran, Janis, Luzi, Mari, Maysa, Michele, Nathi, Renan, Sika e Thais eu só tenho a agradecer por tudo o que eu aprendi nesse tempo em que estive com vocês. Espero conseguir passar a diante todos os ensinamentos que adquiri enquanto vocês todos ainda estavam por aqui. Grupo W, obrigada por estarem sempre me ensinando e dando puxões de orelha. Este trabalho é nosso, porque somos e sabemos o que é ser um GRUPO. Luciene, Marcela e Juliana, vocês foram as pessoas certas no lugar certo. Durante muito tempo eu ainda irei agradecer a Deus por ter colocado vocês na minha vida. Vocês são minha segunda família. Débora, Nayara, Paula, Samara e Cássia, obrigada pela amizade de alguns 20 e poucos anos. Vocês estiveram e estarão presentes nos momen...