Antiaging effects of a skin care formulation containing nanoencapsulated antioxidants: A clinical, in vitro, and ex vivo study.

BACKGROUND: The development of effective cosmetic products for the reduction of the signs of skin aging is a complex process which requires an optimized combination of ingredients and specialized systems to deliver the actives to the skin layers. AIM: To evaluate the tolerance and antiaging clinical efficacy of a cosmetic formulation containing a blend of nanoencapsulated antioxidants: ascorbyl palmitate, resveratrol, tocopherol, caffeine, carnosine, and niacinamide. METHODS: Clinical efficacy was determined by subjective and instrumental analyses of collagen synthesis by fluorescence spectroscopy, by three-dimensional imaging analysis of suborbital edema, and by analysis of skin hydration and sebum content by biophysical techniques-Corneometer® and Sebumeter®. RESULTS: The studied formulation was safe and effective for the improvement of skin appearance by increasing collagen synthesis and skin moisturizing and by reducing facial blemishes, swelling, and oiliness. A preclinical exploratory approach using an experimental model of human cell and skin cultures agreed with the observed antiaging effects, identifying mechanisms related to the containment of oxidative stress, reduction of melanin production, increased synthesis of type I procollagen, and regulation of the epidermal cohesion protein filaggrin. CONCLUSIONS: The skin benefits obtained resulted from the combination of the ingredients in the formulation and the nanoencapsulation-based delivery system, which favors the solubility, safety, efficacy, and bioavailability of the preparation to the skin.

Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

Estudo comparativo para avaliação da eficácia cicatrizante de formulações tópicas contendo Triticum aestivum L. (sinônimo Triticum vulgare) em modelo de pele humana nativa / Comparative study to evaluate the wound healing efficacy of topical formulations containing Triticum aestivum L. (Triticum vulgare) in a native human skin model

Introdução: extratos vegetais e ativos derivados de plantas tem sido desenvolvidos com o objetivo de melhorar e potencializar o processo de cicatrização cutânea, dentre eles, o Triticum aestivum L. (sinônimo Triticum vulgare). Objetivo: avaliar o efeito do extrato de grão inteiro (EGTA-PR) e extrato aquoso (EATA-FI) de Triticum aestivum L. na cicatrização cutânea em pele humana ex vivo. Métodos: fragmentos de pele obtidos de cirurgia plástica eletiva foram submetidos a lesões teciduais e tratados com os extratos durante oito dias para avaliação histológica da reepitelização e marcação proteica do fator de crescimento epidérmico (EGF). Resultados: EGTA-PR e EATA-FI aceleraram o processo de reepitelização em cultura de pele humana submetida a lesão tecidual. Adicionalmente, foi observado um aumento da marcação proteica de EGF após o tratamento com EGTA-PR. Conclusão: EGTA-PR apresentou um melhor desempenho na reepitelização quando comparado ao EATA-FI, pois apresentou uma maior marcação proteica para EGF em cultura de pele humana. Da mesma forma, os resultados histológicos mostraram que a redensificação dérmica obtida com o EGTA-PR foi visualmente superior à observada com EATA-FI. Os resultados obtidos são promissores e corroboram as diversas ações biológicas já reportadas na literatura para extrato de Triticum aestivum L. nas etapas da cicatrização tecidual.

Introduction: Plant extracts and actives derived from plants were developed to improve and enhance the skin healing process including Triticum aestivum L. (Triticum vulgare). Purpose: To evaluate the effect of whole grain extract (EGTA-PR) and aqueous extract (EATA-FI) of Triticum aestivum L., on ex vivo skin healing. Methods: Skin fragments obtained from elective plastic surgery were subjected to tissue damage and treated with extracts for eight days for histological evaluation of re-epithelialization and immunofluorescence for epidermal growth factor (EGF). Results: EGTA-PR and EATA-FI accelerated the re-epithelialization process in human skin culture submitted to tissue injury. Additionally, we observed increased EGF protein labeling after treatment with EGTA-PR. Conclusion: EGTA-PR showed a better performance in re-epithelialization when compared to EATA-FI, as it presented a higher protein labeling for EGF in human skin culture. Likewise, the histological results showed that the dermal redensification obtained with EGTA-PR was visually superior to that observed with EATA-FI. The results obtained are promising and corroborate the several biological actions already reported in the literature for Triticum aestivum L. extract in tissue healing stages

Invitro effect of pine bark extract on melanin synthesis, tyrosinase activity, production of endothelin-1, and PPAR in cultured melanocytes exposed to Ultraviolet, Infrared, and Visible light radiation.

BACKGROUND: French maritime pine bark (Pinus pinaster) extract (PBE), the registered trade name of which is Pycnogenol® , has been studied for its depigmenting action due to its antioxidant, anti-inflammatory, and anti-melanogenic activity. However, the mechanisms through which PBE are still not fully clear. OBJECTIVE: Evaluate the impact of PBE on four in vitro parameters closely associated with cutaneous pigmentation, including melanin synthesis, tyrosinase activity, endothelin-1 (ED1), and production of peroxisome proliferator-activated receptor α, δ, and γ (PPAR α, δ, and γ), by studying the modulation of action of ultraviolet radiation A (UVA)/ultraviolet radiation B (UVB), infrared-A (IR-A), visible light (VL), and association of UVA/UVB, IR-A, and VL (ASS). METHODS: Human melanocytes were incubated in a dry extract solution of PBE, exposed to UVA/UVB, IR-A, VL, and ASS for subsequent quantification of melanin, ED1, and PPAR α, δ, and γ. The effects of PBE on inhibition of tyrosinase activity were also performed by monophenolase activity assay. RESULTS: UVA/UVB, IR-A, VL, and ASS radiation caused significant increases in the synthesis of melanin, ED1, and PPAR α, δ, and γ when compared to baseline control. However, PBE significantly reduced the production of melanin, ED1, and PPAR α, δ, and γ, as well as reducing about 66.5% of the tyrosinase activity. CONCLUSIONS: PBE reduces in vitro melanin production by downregulating tyrosinase and reducing pigmentation-related mediators, such as ED1 and PPAR α, δ, and γ, therefore contributing to the inhibition of pathways associated with skin hyperpigmentation.

Ex Vivo Human Skin: An Alternative Test System for Skin Irritation and Corrosion Assays.

Native human skin has been reported in the literature as being an important experimental model for studying skin biology. Studies performed by our group have shown that ex vivo skin, from elective plastic surgery, maintains the biological characteristics of native skin under specific culture conditions. As such, it might be a feasible model for the safety and efficacy testing of topical substances. While Brazil is at the forefront of global regulation implementation, Brazilian researchers are not always able to transfer certain widely used protocols to their laboratories, particularly protocols that involve the use of reconstructed tissues with limited viability, such as those for skin corrosion (OECD TG 431) and irritation testing (OECD TG 439). In this study, we investigated the applicability of the ex vivo skin model to the evaluation of irritation and corrosion potential of a number of proficiency substances described in TG 431 and TG 439. The skin fragments were standardised in size and diameter, and placed into cell culture inserts. The experimental protocol was conducted according to TG 431 and TG 439. The results obtained show that ex vivo skin could represent a promising tool for the evaluation of irritation and corrosion potential of substances (subject to inclusion and exclusion criteria), as recommended by OECD guidelines. While this is a proof-of-concept study, the use of ex vivo skin should be considered for such testing.

Modulation of skin androgenesis and sebum production by a dermocosmetic formulation.

BACKGROUND: Excessive androgenesis in the skin promotes sebaceous hyperproduction which is the onset of acne vulgaris pathogenesis. Free fatty acids and lipid accumulation in the glandular infundibulum culminates in microbiota imbalance, triggering inflammatory response and follicular hyperkeratinization. AIMS: The purpose of this work was to present an alternative cosmetic treatment for acne skin care, focusing on the prevention of sebaceous gland dysregulation. METHODS: Insulin-stimulated human sebocytes were treated with noncytotoxic concentrations of a DTRW cosmetic formulation. After 6 days of incubation, cell lysates were collected for testosterone, 5α-reductase, and dyhidrotestosterone (DHT) quantitation. In parallel, cells were stained with Oil Red O to measure sebum production. RESULTS: Human sebocytes were incubated with insulin to mimic a seborrheic microenvironment with overproduction of intracellular lipids and fatty acids. Concomitant incubation of cell cultures with DRTW was able to promote a 52.97% reduction in intracellular lipid content. The anti-androgenic properties of DRTW had been proved by the reductions of testosterone (↓59.90%), 5α reductase (↓59.34%), and DHT (↓55.98%) levels in sebocyte cultures also stimulated with insulin. CONCLUSION: The results indicate a promising action of DRTW cosmetic formulation in preventing the development of acne lesions by mechanisms involving the modulation of cutaneous androgenesis and consequently the control of sebum overproduction, considered one of the leading causes of acne.

Novel complex of cosmetic ingredients with promising action in preventing hair loss and follicular aging through mechanism involving enrichment of WNT/signaling, mitochondrial activity, and stem cells maintenance.

BACKGROUND: Mechanisms involved in hair metabolism are diverse, and the availability of ingredients that normalize dysfunctions or mitigate the effects of extrinsic stress suffered daily is greatly desired by consumers to improve the aesthetic appearance of hair. AIMS: In this work, we carried out a preclinical exploratory approach to evaluate the effects of a complex of nanoencapsulated active ingredients (AcPi), as well as a cosmetic formulation containing AcPi (ShPi and HtPi) in mechanisms involving hair loss and follicular aging. METHODS: Human hair follicle dermal papilla cells and human scalp culture were treated with AcPi, ShPi, or HtPi and stimulated with UV radiation or testosterone for further measurement of mitochondrial biogenesis, reactive oxygen species (ROS), ß-catenin, dyhidrotestosterone (DHT), collagen XVIIα1 (COL17A1), and cutaneous permeation. RESULTS: Our results demonstrated that AcPi prevents oxidative stress and balances mitochondial activity disturbed by exposure to UV radiation. AcPi also promoted an enrichment of WNT/ß-catenin signaling pathway, stimulating hair growth, and lengthening the anagen phase of hair cycle. ShPi and HtPi were able to prevent hair aging, minimizing the excessive degradation of COL17A1 in hair follicle exposed to UV radiation, in addition to controlling androgenic metabolism by reducing DHT production. CONCLUSION: The integral effects of AcPi have not been completely elucidated; however, these results, associated with clinical evidences, allow us to infer that this ingredient prevents follicular aging, miniaturization, and consequently hair loss by mechanisms involving energetic homeostasis maintenance, antioxidant, and anti-androgenic actions.

The Ex Vivo Skin Model as an Alternative Tool for the Efficacy and Safety Evaluation of Topical Products.

The development of alternative approaches for safety and efficacy testing that avoid the use of animals is a worldwide trend, which relies on the improvement of current models and tools so that they better reproduce human biology. Human skin from elective plastic surgery is a promising experimental model to test the effects of topically applied products. As the structure of native skin is maintained, including cell population (keratinocytes, melanocytes, Langerhans cells and fibroblasts) and dermal matrix (containing collagen, elastin, glycosaminoglycans, etc.), it most closely matches the effects of substances on in vivo human skin. In this review, we present a collection of results that our group has generated over the last years, involving the use of human skin and scalp explants, demonstrating the feasibility of this model. The development of a test system with ex vivo skin explants, of standard size and thickness, and cultured at the air-liquid interface, can provide an important tool for understanding the mechanisms involved in several cutaneous disorders.

Whitening effects of cosmetic formulation in the vascular component of skin pigmentation.

BACKGROUND: Melanin plays an important role in protecting the skin against the harmful effects of solar radiation, but its abnormal accumulation may become an aesthetic problem, such as melasma and age spots. AIMS: The aim of this study was to evaluate the antiangiogenic and whitening effects of a depigmentation formulation (BLTX) using an in vitro model of human cell and skin culture. METHODS: Human fibroblasts, keratinocytes or melanocytes were treated with BLTX and subjected to oxidative stress by UV radiation or inflammatory stress with IL-1α for quantification of melanin, tyrosinase, endothelin-1, PAR-2, VEGF and iNOS. Fragments of human skin, from elective plastic surgery, were treated with BLTX and subjected to histological evaluation with hematoxylin/eosin associated with Fontana-Masson technique for melanin view. A parametric method, the one-way analysis of variance (ANOVA) followed by the Bonferroni test, was used to compare data among all groups. RESULTS: The results demonstrated that BLTX promotes a reduction in VEGF and iNOS protein synthesis in cultured dermal fibroblasts, indicating an antiangiogenic property. In relation to whitening effect, BLTX was able to reduce the production of melanin in both systems, melanocytes and human skin cultures. The depigmenting action was also revealed by decreasing the levels of endothelin-1, PAR-2 and activity of tyrosinase, when compared to cultures exposed to UV radiation. CONCLUSION: The results allow us to infer that BLTX presents an antiangiogenic effect indicating a role in the vascular component of melasma. Furthermore, the whitening property observed reinforces its use in the prevention and treatment of melasma.

Avaliação pré-clínica dos efeitos profiláticos do extrato de Pinus pinaster (Pycnogenol®) sobre a deposição cutânea de hemossiderina / Pre-clinical evaluation of the profilatic effects of Pinus pinaster extract (Pycnogenol®) on skin hemosiderin deposits

Introdução: A escleroterapia é o método mais utilizado para o tratamento de varizes dos membros inferiores tendo como complicação mais comum o aparecimento de manchas hipercrômicas na região tratada. O Pycnogenol® é conhecido há muito tempo como um flebotônico, anti-inflamatório e despigmentante da pele. Estudos já comprovaram a eficácia deste fármaco na prevenção e no tratamento da hiperpigmentação pós-inflamatória. Objetivo: Avaliar a eficácia do extrato de Pinus pinaster (Pycnogenol®; EPP) na prevenção de depósitos de hemossiderina em cultura de pele humana submetida a estresse inflamatório. Métodos: Fragmentos de pele humana foram estimulados com interleucina 1 alfa (IL-1a) para indução de uma resposta inflamatória e, concomitantemente, tratados com EPP para posterior avaliação histológica e semi-quantificação de hemossiderina. Resultados: A avaliação histológica dos fragmentos de pele expostos à IL-1alfa; revelaram uma densidade de hemossiderina 26,6% maior em comparação ao grupo controle. Por outro lado, os fragmentos de pele incubados concomitantemente com EPP mostraram reduções significativas na deposição de hemossiderina quando comparados ao grupo somente expostos ao microambiente inflamatório. Conclusões: Os resultados apresentados neste estudo apontam para um importante efeito do EPP (Pycnogenol®) na prevenção do acúmulo de hemossiderina originado pelo estresse inflamatório semelhante ao processo pós escleroterapia.

Introduction: Sclerotherapy is the most widely used method for the treatment of varicose veins of the lower limbs. The most common complication is the appearance of hyperchromic spots in the treated region. Pycnogenol® has long been known as a phlebotonic, anti-inflammatory and skin depigmenting drug. Studies have already proven the efficacy of this drug in the prevention and treatment of post inflammatory hyperpigmentation. Objective: To evaluate the effectiveness of Pinus pinaster extract (Pycnogenol®; PPE) in the prevention of hemosiderin deposits in human skin culture submitted to inflammatory stress. Methods: Fragments of human skin were stimulated with interleukin 1 alpha (IL-1 a) to induce an inflammatory response and, concurrently, treated with PPE for further histological evaluation and hemosiderin semi-quantification. Results: The histological evaluation of skin fragments exposed to IL-1 alpha revealed a 26.6% higher hemosiderin density compared with the control group. Moreover, skin fragments incubated concomitantly with PPE showed significant reductions in hemosiderin deposits when compared with the group only exposed to the inflammatory microenvironment. Conclusions: The results presented in this study showed an important effect of PPE (Pycnogenol®) in the prevention of hemosiderin accumulation caused by inflammatory stress similar to the post-sclerotherapy process.

Sterol-standardized phytopharmaceutical from ground cherry: Corticoid-like properties on human keratinocytes and fibroblasts and its effects in a randomized double-blind placebo-controlled clinical trial.

BACKGROUND: Topical corticosteroids have been the most commonly prescribed drugs to treat skin inflammation, but their uses can lead to several adverse effects. Nowadays, new pharmacological strategies have been evaluated to improve dermatologic efficacy and reduce adverse effects, including natural products. OBJECTIVES: The aim of this study was to evaluate and compare the effects of a plant sterol standardized supercritical CO2 phytopharmaceutical of Physalis angulata L. with hydrocortisone on the immune and inflammatory mediators, and skin repair components production. Moreover, we studied effects of both products on the skin microcirculation and temperature in a double-blind placebo-controlled clinical trial. METHODS: Both products were evaluated on the immune (IL-6, IL-10, INF-γ, TNF-α, and IL-1α), inflammatory (COX-2, LOX, PLA2 , PGE2 , LTB4 , histamine, and NF-κB), and repair components (TGF-ß, GM-CSF, collagen, and GAG) production on human keratinocytes and fibroblast in non-stimulated and LPS-stimulated conditions. Indeed, in a randomized double-blind placebo-controlled clinical trial, we evaluated the effects of the both creams on the skin microcirculation and temperature using laser Doppler and infrared thermometer, respectively. RESULTS: Physalis angulata acted on the skin, modulating immune status and inflammatory response producing corticoid-like effects, but different of hydrocortisone, increased skin repair factors. The effects of phytopharmaceutical cream in the clinical trial promoted a better reduction in skin microcirculation and temperature than hydrocortisone. CONCLUSIONS: Taken together, the results indicate that sterol standardized CO2 supercritical preparation of P angulata is a new and innovative phytopharmaceutical with multiple pharmacological effects potentially useful as human skin protective product, particularly against cutaneous inflammatory disorders.

Toxic effects of phytol and retinol on human glioblastoma cells are associated with modulation of cholesterol and fatty acid biosynthetic pathways.

Glioblastoma (GBM) is the most common primary brain tumor. Genetic mutations may reprogram the metabolism of neoplastic cells. Particularly, alterations in cholesterol and fatty acid biosynthetic pathways may favor biomass synthesis and resistance to therapy. Therefore, compounds that interfere with those pathways, such as phytol (PHY) and retinol (RET), may be appropriate for cytotoxic approaches. We tested the effect of PHY or RET on the viability of human GBM cell lines (U87MG, A172 and T98G). Since the compounds showed a dose-dependent cytotoxic effect, additional analyses were performed with IC50 values. Transcriptome analyses of A172 cells treated with PHY IC50 or RET IC50 revealed down-regulated genes involved in cholesterol and/or fatty acid biosynthetic pathways. Thus, we investigated the expression of proteins required for cholesterol and/or fatty acid synthesis after treating all lineages with PHY IC50 or RET IC50 and comparing them with controls. Sterol regulatory element-binding protein 1 (SREBP-1) expression was reduced by PHY in U87 and T98G cells. However, fatty acid synthase (FAS) protein expression, which is regulated by SREBP-1, was down-regulated in all lineages after both treatments. Moreover, farnesyl-diphosphate farnesyltransferase (FDFT1) levels, a protein associated with cholesterol synthesis, were reduced in all lineages by PHY and in U87MG and A172 cells by RET. Our results suggest that SREBP-1, FAS and FDFT1 are potential target(s) for future in vivo approaches against GBM and support the use of inhibitors of their synthesis, including PHY and RET, for such approaches.

Ultraviolet A photosensitivity profile of dexchlorpheniramine maleate and promethazine-based creams: Anti-inflammatory, antihistaminic, and skin barrier protection properties.

BACKGROUND: Unwanted side effects such as dryness, hypersensitivity, and cutaneous photosensitivity are challenge for adherence and therapeutical success for patients using treatments for inflammatory and allergic skin response. AIMS: In this study, we compared the effects of two dermatological formulations, which are used in inflammatory and/or allergic skin conditions: dexchlorpheniramine maleate (DCP; 10 mg/g) and promethazine (PTZ; 20 mg/g). METHODS: We evaluated both formulations for phototoxicity potential, skin irritation, anti-inflammatory and antihistaminic abilities, and skin barrier repair in vitro and ex vivo using the standard OECD test guideline n° 432, the ECVAM protocol n° 78, and cultured skin explants from a healthy patient. Ultraviolet A was chosen as exogenous agent to induce allergic and inflammatory response. RESULTS: Both PTZ and DCP promoted increases in interleukin-1 (IL-1) synthesis in response to ultraviolet A (UVA) radiation compared to control. However, the increase observed with PTZ was significantly greater than the DCP, indicating that the latter has a lower irritant potential. DCP also demonstrated a protective effect on UVA-induced leukotriene B4 and nuclear factor kappa B (NF-κB) synthesis. Conversely, PTZ demonstrates more robust UVA antihistaminic activity. Likewise, PTZ promoted a significantly greater increase in the production of involucrin and keratin 14, both associated with protective skin barrier property. CONCLUSION: In conclusion, these data suggest possible diverging UVA response mechanisms of DCP and PTZ, which gives greater insight into the contrasting photosensitizing potential between DCP and PTZ observed in the patients.

Honokiol protects skin cells against inflammation, collagenolysis, apoptosis, and senescence caused by cigarette smoke damage.

BACKGROUND: Pollution, especially cigarette smoke, is a major cause of skin damage. OBJECTIVES: To assess the effects of the small molecule polyphenol, honokiol, on reversing cigarette smoke-induced damage in vitro to relevant skin cells. METHODS: Keratinocytes (HaCat) cultures were exposed to cigarette smoke and, after 48 hours, IL-1α and IL-8 were measured in cell supernatants. Moreover, TIMP-2 production, apoptosis rate, and senescence ß-galactosidase expression were evaluated in primary human foreskin fibroblasts (HFF-1) cultures. RESULTS: Honokiol at 10 µm reduced IL-1α production by 3.4 folds (P < 0.05) and at 10 and 20 µm reduced IL-8 by 23.9% and 53.1% (P < 0.001), respectively, in HaCat keratinocytes. In HFF-1, honokiol restored TIMP-2 production by 96.9% and 91.9% (P < 0.001), respectively, at 10 and 20 µm, as well as reduced apoptosis by 47.1% (P < 0.001) and 41.3% (P < 0.01), respectively. Finally, honokiol reduced senescence-associated ß-galactosidase expression in HFF-1. CONCLUSION: Honokiol protects both HFF-1 and HaCat against cigarette smoke-induced inflammation, collagenolysis, apoptosis, and senescence.

Characteristics of sulfasalazine-induced cytotoxicity in C6 rat glioma cells.

Glioblastoma is the most aggressive primary brain tumor. Surgical resection, radiotherapy and temozolomide (TMZ), an alkylating agent, is the standard of care. Glioma cells may synthetize the antioxidant glutathione by importing cystine through a cystine/glutamate antiporter, which is inhibited by sulfasalazine (SAS). C6 rat glioma cells are largely used in in vitro and in vivo models for developing new glioblastoma treatment strategies. We treated C6 cells with 25µM TMZ and/or 0.25mM or 0.5mM SAS for 1, 3 or 5days and evaluated viability, apoptosis, total glutathione levels and metalloproteinase MMP2 and MMP9 activities. TMZ treatment slightly reduced cell viability by 9.5% compared with vehicle treatment (0.1% dimethyl sulfoxide) only after 5days. In addition, TMZ did not modify apoptosis, glutathione content or MMP2/MMP9 activities. The 0.25mM SAS treatment reduced cell viability by 31.1% and 19.4% after the first and third days, respectively. This effect was not sustained after the fifth day of treatment. In contrast, 0.5mM SAS caused a reduction in cell viability by nearly 100%, total glutathione depletion and apoptosis induction. Moreover, the effect of 0.5mM SAS was greater than that of TMZ in terms of cell viability reduction, total glutathione depletion and apoptosis induction. MMP9 activity was reduced by 40% after 5days of 25µM TMZ and 0.5mM SAS co-administration. Considering previous data from our group, we verified that the cellular viability results differed between rat and human cells; C6 cells were more vulnerable to 0.5mM SAS than human A172 and T98G glioblastoma lineages. We propose that C6 cells may not be appropriate for studying human glioblastoma and that the results obtained using these cells should be interpreted with caution.

Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells.

Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X c (-) , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25 µM) and/or 0.5 mM SAS for 1, 3, or 5 days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5 days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5 days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 cells.

Metodologia alternativa para o estudo dos efeitos da radiação infravermelha-A sobre a pele humana / Alternative methodology for the study of infrared-A radiation effects on human skin

Introdução: A radiação infravermelha A (IV-A) causa alterações estruturais na pele, similares àquelas provocadas pela exposição prolongada à radiação ultravioleta. A avaliação de eficácia e segurança para produtos cosméticos concentra-se em ensaios in vitro e clínicos. Uma alternativa promissora é a utilização de fragmentos de pele humana provenientes de cirurgias plasticas eletivas, para avaliar os reais beneficios os reais benefícios clínicos de um produto aplicado topicamente. Objetivo: O objetivo desta investigação foi correlacionar os efeitos da radiação IV-A, em biópsias e em fragmentos de pele ex vivo e cultura de fibroblastos humanos, pela quantificação dos mediadores MMP-1, TIMP-1 e GADD45a. Métodos: Coleta de biópsias de 15 voluntárias após aplicações de IV-A durante cinco dias consecutivos. Exposição à radiação IV-A de fragmentos de pele humana provenientes de cirurgia plástica eletiva e cultura de fibroblastos humanos. Mensuração dos mediadores MMP-1, TIMP-1 e GADD45a para posterior comparação dos resultados. Resultados: Nos três modelos utilizados a radiação IV-A induziu aumento de MMP-1, inibiu a síntese de GADD45a e não alterou os valores de TIMP-1. Conclusão: Devido à correlação positiva dos modelos estudados, pode-se sugerir o uso de pele ex vivo como ferramenta plausível e sustentável para suprir diferenças entre conhecimentos gerados a partir de experimentos in vitro e clínico.

Introduction: Infrared radiation (IR-A) causes structural changes in the skin, similar to those caused by prolonged exposure to ultraviolet radiation. Evaluation of efficacy and safety of cosmetic products concentrates in in vitro tests and clinical trials. A promising alternative is the use of fragments of human skin from elective cosmetic surgery, to evaluate the actual clinical benefits of a product applied topically. Objective: The objective of this study was to correlate IR-A radiation effects in biopsies and in ex vivo skin fragments and in human fibroblasts culture by quantifying MMP-1, TIMP-1 and GADD45a mediators. Methods: Collection of biopsies from 15 volunteers after IR-A applications for 5 consecutive days. Exposure to IR-A radiation of human skin fragments from elective cosmetic surgery, and human fibroblasts culture. Measurement of MMP-1, TIMP-1 and GADD45a mediators for further comparison of results. Results: In the three models used, the IR-A radiation induced an increase in MMP-1, inhibited the synthesis of GADD45a, and did not changed TIMP-1 values. Conclusion: Due to the positive correlation of the models studied, it may be suggested the use of ex vivo skin as plausible and sustainable tool to overcome differences between knowledge generated from in vitro and clinical experiments.

Antiageing Mechanisms of a Standardized Supercritical CO 2 Preparation of Black Jack (Bidens pilosa L.) in Human Fibroblasts and Skin Fragments.

The use of topical retinoids to treat skin disorders and ageing can induce local reactions, while oral retinoids are potent teratogens and produce several unwanted effects. This way, efforts to explore complementary care resources should be supported. Based on this, we evaluate the antiageing effects of a supercritical CO2 extract from Bidens pilosa L. (BPE-CO2A) containing a standardized multicomponent mixture of phytol, linolenic, palmitic, linoleic, and oleic acids. BPE-CO2A was assessed for its effects on human dermal fibroblasts (TGF-ß1 and FGF levels using ELISA; collagen, elastin, and glycosaminoglycan by colorimetric assays, and mRNA expression of RXR, RAR, and EGFr by qRT-PCR) and human skin fragments (RAR, RXR, collagen, elastin, and glycosaminoglycan by immunohistochemical analysis). Levels of extracellular matrix elements, TGF-ß1 and FGF, and EGFr gene expression were significantly increased by BPE-CO2A. The modulation of RXR and RAR was positively demonstrated after the treatment with BPE-CO2A or phytol, a component of BPE-CO2A. The effects produced by BPE-CO2A were similar to or better than those produced by retinol and retinoic acid. The ability to stimulate extracellular matrix elements, increase growth factors, and modulate retinoid and rexinoid receptors provides a basis for the development of preparation containing BPE-CO2A as an antiageing/skin-repair agent.

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis.

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to naïve control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the naïve ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.

Violacein extracted from Chromobacterium violaceum inhibits Plasmodium growth in vitro and in vivo.

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.