Silent peripheral neuropathy determined by high-resolution ultrasound among contacts of patients with Hansen's disease

IntroductionHansen's disease (HD) primarily infects peripheral nerves, with patients without HD being free of peripheral nerve damage. Household contacts (HHCs) of patients with HD are at a 5–10 times higher risk of HD than the general population. Neural thickening is one of the three cardinal signs that define a case of HD according to WHO guidelines, exclusively considering palpation examination that is subjective and may not detect the condition in the earliest cases even when performed by well-trained professionals. High-resolution ultrasound (HRUS) can evaluate most peripheral nerves, a validated technique with good reproducibility allowing detailed and accurate examination.ObjectiveThis study aimed to use the peripheral nerve HRUS test according to the HD protocol as a diagnostic method for neuropathy comparing HHCs with healthy volunteers (HVs) and patients with HD.MethodsIn municipalities from 14 different areas of Brazil we selected at random 83 HHC of MB-patients to be submitted to peripheral nerve ultrasound and compared to 49 HVs and 176 HD-patients.ResultsHousehold contacts assessed by HRUS showed higher median and mean absolute peripheral nerve cross-sectional area (CSA) values and greater asymmetries (ΔCSA) compared to HVs at the same points. Median and mean absolute peripheral nerve CSA values were higher in patients with HD compared to HCCs at almost all points, while ΔCSA values were equal at all points. Mean ± SD focality (ΔTpT) values for HHCs and patients with HD, respectively, were 2.7 ± 2.2/2.6 ± 2.2 for the median nerve, 2.9 ± 2.7/3.3 ± 2.9 for the common fibular nerve (p > 0.05), and 1.3 ± 1.3/2.2 ± 3.9 for the ulnar nerve (p < 0.0001).DiscussionConsidering HRUS findings for HHCs, asymmetric multiple mononeuropathy signs (thickening or asymmetry) in at least 20% of the nerves evaluated could already indicates evidence of HD neuropathy. Thus, if more nerve points are assessed in HHCs (14 instead of 10), the contacts become more like patients with HD according to nerve thickening determined by HRUS, which should be a cutting-edge tool for an early diagnosis of leprosy cases

Efficacy of natural rubber latex serum from Hevea brasiliensis in skin abrasion healing in rats

Quando as escoriações ocorrem é esperada uma cicatrização sem complicações, utilizando produtos para prevenção de infecções e que proporcionam ambiente que otimiza rápida cicatrização com cicatrizes mínimas. Tratamentos com antissépticos tópicos e produtos cicatrizantes são os mais utilizados. Vários relatos demonstram que o soro do látex (SLX) da seringueira Hevea brasiliensis tem propriedades cicatrizantes para úlceras cutâneas. O objetivo deste trabalho foi avaliar citotoxicidade e potencial proliferativo do SLX em ensaios in vitro e sua eficácia em escoriações cutâneas no dorso de ratos. A citotoxicidade do SLX (10%, 1% e 0,1%) foi avaliada em culturas de fibroblastos 3T3 pela determinação da viabilidade celular. A proliferação/migração celular com queratinócitos humanos foi avaliada pelo método scratch assay nas concentrações (1%, 0,1%, 0,01%, 0,001%, 0,0001% e 0,00001%). Para a atividade cicatrizante utilizou-se 72 ratos Wistar submetidos à escoriação no dorso por dermoabrasão, e 3 grupos foram estabelecidos: soro fisiológico (SF), antisséptico (AS-Merthiolate®) e soro do látex (SLX-Regederm®), aplicado diariamente por 10 dias. As lesões foram fotografadas e avaliadas por análise de imagem nos dias 2, 7 e 10 póslesão e calculados os índices de cicatrização das escoriações (ICE) para medir reepitelização. Nos dias 2, 7 e 10 foram sacrificados 8 animais de cada grupo e coletadas amostras para dosagem bioquímica da mieloperoxidase (MPO) e no 10º dia uma parte das biópsias foram separadas para análise histológica da epiderme e crosta. O SLX não apresentou toxicidade aos fibroblastos nas concentrações menores ou iguais à 1%, sendo inviável na concentração de 10%. Pelo scratch assay o SLX apresentou atividade proliferativa sobre queratinócitos humanos principalmente na concentração de 0,01% (89%). O SLX promoveu melhor reepitelização no modelo de escoriação, principalmente no 7º dia, diferente dos demais grupos que tinham atraso na reepitelização. Além disso, quanto às diferenças de espessura da crosta e da epiderme, o grupo SLX promoveu um aumento no número de camadas epidérmicas e menor espessura das crostas. Os grupos SLX e AS apresentaram aumento da enzima MPO no 2º dia, porém com redução significante a partir do 7º dia, diminuindo assim a inflamação e acelerando consequentemente a reepitelização. Os resultados evidenciaram que a aplicação do soro do látex é segura pela não toxicidade e comprova sua eficácia perante a outros produtos, pela propriedade proliferativa para queratinócitos e pela aceleração da cicatrização em modelos de exulcerações cutâneas.When abrasions occur is expected to heal without complications, using products to prevent infections and provide environment that optimizes rapid healing with minimal scarring. Treatments with topical antiseptics and healing products are the most used. Several reports show that the latex serum (LXS) from Hevea brasiliensis has healing properties for skin ulcers. The objective was to evaluate the cytotoxic and proliferative potential of LXS in vitro assays and their efficacy to heal skin abrasions on the back of rats. LXS cytotoxicity (10, 1 and 0.1%) was evaluated in 3T3 fibroblast cultures by cell viability. The cell proliferation/migration in human keratinocytes was evaluated by the scratch assay method using 1%, 0.1%, 0.01%, 0.001%, 0.0001% and 0.00001% concentrations. For healing activity, 72 Wistar rats were underwent to dermoabrasion on the back and 3 groups were stablished: saline (S), antiseptic (AS-Merthiolate®) and rubber latex serum (LXS-Regederm®). The different products were applied daily for 10 days. The lesions were photographed and evaluated by image analysis on days 2, 7 and 10 post-injury and the abrasion healing rate (AHR) were calculated for evaluation of the re-epithelialization. On days 2, 7 and 10, 8 animals from each group were sacrificed, and samples were taken for biochemical determination of myeloperoxidase (MPO) After that, on day 10, a part of the biopsies was separated for histological analysis of the epidermis and crust. The LXS showed no toxicity to fibroblasts at concentrations less than or equal 1%, being impractical at a concentration of 10%. By the scratch assay, the LXS showed proliferative activity on human keratinocytes, particularly at a concentration of 0.01% (89%). The LXS produced better re-epithelialization in the excoriation model, especially on the 7th day, unlike other groups, in which the re-epithelialization was delayed. Furthermore, it showed significant differences regarding the amount of epidermis and crust. The Regederm group presented an increase in the number of epidermal layers and thinner thickness of their crusts. The LXS and AS groups showed an increase of MPO enzyme on the 2nd day, however, with a significant decrease from the 7th day, thus reducing inflammation and consequently accelerating re-epithelialization. The results showed that the application of the rubber latex serum is safe due its non-toxicity and prove its efficacy comparing to other products, because of its proliferative property for keratinocytes and acceleration of healing in cutaneous exulceration models

Validation of the ex vivo model of human skin (hOSEC) for pharmaceutical ingredient absorption assay

Métodos alternativos ao uso de animais vêm sendo estudados e desenvolvidos ao longo dos anos, merecendo destaque a cultura de explante de pele organotípica humana (hOSEC). Considerada o modelo que mais se aproxima da pele humana em condições in vitro, apresenta estrutura 3D e todas as células da pele nativa, sendo um modelo versátil para o estudo de várias doenças e testes de novos fármacos. Nesse contexto, o objetivo deste trabalho foi validar o modelo ex vivo de pele humana (hOSEC) para ensaio de absorção de insumos farmacêuticos. Foram utilizados os fármacos Dacarbazina e Rifampicina. A viabilidade celular para ambos os fármacos foi testada pelo ensaio MTT em queratinócitos e fibroblastos primários e em linhagens imortalizadas da pele (HaCaT e 3T3). Também foi testada a sensibilidade de linhagens de melanoma humano A375, 1205 Lu e SK-MEL-103 tratadas com Dacarbazina nos tempos de 24, 48 e 72 horas. A viabilidade tecidual do modelo hOSEC tratado com os fármacos foi avaliada pelo método TTC nos tempos de 24, 48, 72 e 96 horas. A Dacarbazina e o modelo hOSEC foram validados por meio de cromatografia líquida de alta eficiência e, seguida à validação, a absorção do fármaco pelo tecido nos tempos de 30 minutos, 1, 3, 6, 12 e 24 horas foi analisada também por cromatografia. A estrutura da pele e apoptose pós-tratamento com Dacarbazina foram analisados por histologia corada com hematoxilina e eosina e TUNEL, respectivamente. Foi realizada a inoculação da linhagem A375 no modelo hOSEC e avaliada o local de aplicação e características das células por meio da coloração hematoxilina e eosina. Os testes de viabilidade frente à Dacarbazina mostraram que tanto nas células primárias quanto nas imortalizadas da pele foram consideradas viáveis (acima de 77%). Em relação as linhagens neoplásicas, a A375 foi a que se mostrou mais sensível em todas as concentrações, principalmente no tempo de 72 horas, diferentemente da 1205 Lu e SK-MEL-103, que se mostraram mais resistentes. Já utilizando a Rifampicina, foi observado uma sensibilidade maior quando exposta à concentração de 200 µg/mL nas quatro diferentes células estudadas. A validação do método foi linear, preciso e exato, demonstrando uma alta confiança para sua utilização. Em relação a absorção da Dacarbazina pela pele, foi observado que a concentração do fármaco na pele foi aumentando e consequentemente diminuindo no meio de cultura até 24 horas, atingindo concentração máxima pela pele de 36,36 µg/mL (18,18%) da dose inicial de 200 µg/mL no tempo de 12 horas. Dados histológicos mostraram que as camadas da pele foram mantidas estruturalmente em todos os tempos estudados. Não foram observadas células apoptóticas nas camadas epidérmica e dérmica. A inoculação das células A375 foi realizada de maneira adequada, intradermicamente, e as características de células neoplásicas foram mantidas. Portanto, nossos resultados confirmam que o modelo hOSEC pode ser um modelo alternativo para avaliar a dinâmica de absorção e distribuição de insumos farmacêuticos na pele, assim como também pode ser possível obter um modelo ex vivo de tumor na pele a partir da inoculação de células neoplásicas.Alternative methods to the use of animals have been studied and developed over the years, with emphasis on the human organotypic skin explant culture (hOSEC). Considered the closest model to human skin under in vitro conditions, it has a 3D structure and all native skin cells, making it a versatile model for the study of various diseases and testing of new drugs. In this context, the aim of this work was to validate the ex vivo model of human skin (hOSEC) for pharmaceutical ingredient absorption assay. The drugs Dacarbazine and Rifampicin were used. Cell viability for both drugs was tested by MTT assay in primary keratinocytes and fibroblasts and in immortalized skin cell lines (HaCaT and 3T3). The sensitivity of human melanoma cell lines A375, 1205 Lu and SK-MEL-103 treated with Dacarbazine was also tested at times of 24, 48 and 72 hours. Tissue viability of the drug-treated hOSEC model was evaluated by the TTC method at times of 24, 48, 72 and 96 hours. Dacarbazine and the hOSEC model were validated using high-performance liquid chromatography and, after validation, the drug absorption by the tissue at times of 30 minutes, 1, 3, 6, 12 and 24 hours was also analyzed by chromatography. Skin structure and apoptosis after treatment with Dacarbazine were analyzed by histology stained with hematoxylin and eosin and TUNEL, respectively. Inoculation of the A375 cell in the hOSEC model was performed and the application site and cell characteristics were evaluated by hematoxylin and eosin staining. Viability tests in relation to Dacarbazine showed that both primary and immortalized skin cells were considered viable (above 77%). Regarding neoplastic cell lines, A375 was the most sensitive at all concentrations, especially at 72 hours, unlike 1205 Lu and SK-MEL-103, which were more resistant. With the use of Rifampicin, a greater sensitivity was observed when exposed to a concentration of 200 µg/mL in the four different cell lines studied. The method validation was linear, precise and exact, demonstrating a high confidence for its use. Regarding the absorption of Dacarbazine through the skin, it was observed that the drug concentration in the skin increased and consequently decreased in the culture medium for up to 24 hours, reaching a maximum concentration through the skin of 36.36 µg/mL (18.18%) of the initial dose of 200 µg/ml in 12 hours. Histological data showed that the skin layers were structurally maintained at all times studied. Apoptotic cells were not observed in the epidermal and dermal layers. Inoculation of A375 cells was adequately performed, intradermally, and the characteristics of neoplastic cells were maintained. Therefore, our results confirm that the hOSEC model may be an alternative model to assess the dynamics of absorption and distribution of pharmaceutical ingredients in the skin, as well as it may also be possible to obtain an ex vivo model of tumor of the skin from the inoculation of neoplastic cells

Chitosan‐alginate membranes accelerate wound healing

The purpose of this study was to evaluate the efficacy of chitosan‐alginate membrane to accelerate wound healing in experimental cutaneous wounds. Two wounds were performed in Wistar rats by punching (1.5 cm diameter), treated with membranes moistened with saline solution (CAM group) or with saline only (SL group). After 2, 7, 14, and 21 days of surgery, five rats of each group were euthanized and reepithelialization was evaluated. The wounds/scars were harvested for histological, flow cytometry, neutrophil infiltrate, and hydroxyproline analysis. CAM group presented higher inflammatory cells recruitment as compared to SL group on 2nd day. On the 7th day, CAM group showed higher CD11b+ level and lower of neutrophils than SL group. The CAM group presented higher CD4+ cells influx than SL group on 2nd day, but it decreased during the follow up and became lower on 14th and 21st days. Higher fibroplasia was noticed on days 7 and 14 as well as higher collagenesis on 21st in the CAM group in comparison to SL group. CAM group showed faster reepithelialization on 7th day than SL group, although similar in other days. In conclusion, chitosan‐alginate membrane modulated the inflammatory phase, stimulated fibroplasia and collagenesis, accelerating wound healing process in rats103510131022COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Chitosan-alginate membranes accelerate wound healing

The purpose of this study was to evaluate the efficacy of chitosan-alginate membrane to accelerate wound healing in experimental cutaneous wounds. Two wounds were performed in Wistar rats by punching (1.5 cm diameter), treated with membranes moistened with saline solution (CAM group) or with saline only (SL group). After 2, 7, 14, and 21 days of surgery, five rats of each group were euthanized and reepithelialization was evaluated. The wounds/scars were harvested for histological, flow cytometry, neutrophil infiltrate, and hydroxyproline analysis. CAM group presented higher inflammatory cells recruitment as compared to SL group on 2(nd) day. On the 7(th) day, CAM group showed higher CD11b(+) level and lower of neutrophils than SL group. The CAM group presented higher CD4(+) cells influx than SL group on 2(nd) day, but it decreased during the follow up and became lower on 14(th) and 21(st) days. Higher fibroplasia was noticed on days 7 and 14 as well as higher collagenesis on 21(st) in the CAM group in comparison to SL group. CAM group showed faster reepithelialization on 7(th) day than SL group, although similar in other days. In conclusion, chitosan-alginate membrane modulated the inflammatory phase, stimulated fibroplasia and collagenesis, accelerating wound healing process in rats. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1013-1022, 2015.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Lipoxin A4 encapsulated in PLGA microparticles accelerates wound healing of skin ulcers.

Lipoxin A4 (LXA4) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA4 in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA4-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA4-MS, unloaded microparticles (Un-MS), soluble LXA4, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA4-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA4, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA4-MS reduced IL-1β and TNF-α, but increased TGF-β, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA4-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (MMP8) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA4-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA4 receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA4 must interact with ALX to induce wound healing. Our results show that LXA4-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers