Jussara Rehder

Model for human skin reconstructed in vitro composed of associated dermis and epidermis

CONTEXT AND OBJECTIVE The technique of obtaining human skin with dermis and epidermis reconstructed from cells isolated from patients can enable autologous skin grafting on patients with few donor sites. It also enables in vitro trials on chemicals and drugs. The objective of this work was to demonstrate a method for obtaining human skin composed of associated dermis and epidermis, reconstructed in vitro. DESIGN AND SETTING Experimental laboratory study, in the Skin Cell Culture Laboratory of Faculdade de Ciências Médicas, Universidade Estadual de Campinas. METHODS Cells from human fibroblast cultures are injected into bovine collagen type I matrix and kept immersed in specific culturing medium for fibroblasts. This enables human dermis reconstruction in vitro. On this, by culturing human keratinocytes and melanocytes, differentiated epidermis is formed, leading to the creation of human skin composed of associated dermis and epidermis, reconstructed in vitro. RESULTS We showed that human skin composed of associated dermis and epidermis can be successfully reconstructed in vitro. It is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with cells and extracellular matrix organized in parallel to multilayer epidermis. CONCLUSIONS It is possible to obtain completely differentiated human skin composed of associated dermis and epidermis, reconstructed in vitro, from injection of human fibroblasts into bovine collagen type I matrix and culturing of human keratinocytes and melanocytes on this matrix.

Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis

CONTEXT Recent progress in the field of epithelial culture techniques has allowed the development of culture systems in which the reconstructed epidermis presents characteristics of morphological differentiation similar to those seen in vivo. Human epidermis reconstructed in vitro may be used as the best alternative for the in vitro testing of the toxicology and efficiency of products for topical use, as well as in the treatment of skin burns and chronic skin ulcers. OBJECTIVE To demonstrate a method for obtaining human epidermis reconstructed in vitro, using keratinocytes and melanocytes cultivated on dead de-epidermized human dermis. TYPE OF STUDY Experimental/laboratory. SETTING Skin Cell Culture Laboratory of the Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. PROCEDURE Human keratinocytes and melanocytes cultured in vitro were grown on a biological matrix (dead de-epidermized human dermis) and the system was kept at an air-liquid interface, in a suitable culturing medium, until a stratified human epidermis was formed, maintaining the histological characteristics of the epidermis in vivo. RESULTS It was histologically demonstrated that it is possible to reproduce a differentiated epidermis through keratinocytes and melanocytes cultured on dead de-epidermized human dermis, thus obtaining a correctly positioned human epidermis reconstructed in vitro with functional keratinocytes and melanocytes that is similar to in vivo epidermis. CONCLUSIONS It is possible to obtain a completely differentiated human epidermis reconstructed in vitro from keratinocyte and melanocyte cultures on a dead de-epidermized human dermis.

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

High‐Performance Liquid Chromatography Under Partially Denaturing Conditions (dHPLC) is a Fast and Cost‐Effective Method for Screening Molecular Defects: Four Novel Mutations Found in X‐Linked Chronic Granulomatous Disease

Implementing precise techniques in routine diagnosis of chronic granulomatous disease (CGD), which expedite the screening of molecular defects, may be critical for a quick assumption of patient prognosis. This study compared the efficacy of single‐strand conformation polymorphism analysis (SSCP) and high‐performance liquid chromatography under partially denaturing conditions (dHPLC) for screening mutations in CGD patients. We selected 10 male CGD patients with a clinical history of severe recurrent infections and abnormal respiratory burst function. gDNA, mRNA and cDNA samples were prepared by standard methods. CYBB exons were amplified by PCR and screened by SSCP or dHPLC. Abnormal DNA fragments were sequenced to reveal the nature of the mutations. The SSCP and dHPLC methods showed DNA abnormalities, respectively, in 55% and 100% of the cases. Sequencing of the abnormal DNA samples confirmed mutations in all cases. Four novel mutations in CYBB were identified which were picked up only by the dHPLC screening (c.904 insC, c.141+5 g>t, c.553 T>C, and c.665 A>T). This work highlights the relevance of dHPLC, a sensitive, fast, reliable and cost‐effective method for screening mutations in CGD, which in combination with functional assays assessing the phagocyte respiratory burst will contribute to expedite the definitive diagnosis of X‐linked CGD, direct treatment, genetic counselling and to have a clear assumption of the prognosis. This strategy is especially suitable for developing countries.

Immunoarchitectural characterization of a human skin model reconstructed in vitro

CONTEXT AND OBJECTIVE Over the last few years, different models for human skin equivalent reconstructed in vitro (HSERIV) have been reported for clinical usage and applications in research for the pharmaceutical industry. Before release for routine use as human skin replacements, HSERIV models need to be tested regarding their similarity with in vivo skin, using morphological (architectural) and immunohistochemical (functional) analyses. A model for HSERIV has been developed in our hospital, and our aim here was to further characterize its immunoarchitectural features by comparing them with human skin, before it can be tested for clinical use, e.g. for severe burns or wounds, whenever ancillary methods are not indicated. DESIGN AND SETTING Experimental laboratory study, in the Skin Cell Culture Laboratory, School of Medical Sciences, Universidade Estadual de Campinas. METHODS Histological sections were stained with hematoxylin-eosin, Massons trichrome for collagen fibers, periodic acid-Schiff reagent for basement membrane and glycogen, Weigert-Van Gieson for elastic fibers and Fontana-Masson for melanocytes. Immunohistochemistry was used to localize cytokeratins (broad spectrum of molecular weight, AE1/AE3), high molecular weight cytokeratins (34betaE12), low molecular weight cytokeratins (35betaH11), cytokeratins 7 and 20, vimentin, S-100 protein (for melanocytic and dendritic cells), CD68 (KP1, histiocytes) and CD34 (QBend, endothelium). RESULTS Histology revealed satisfactory similarity between HSERIV and in vivo skin. Immunohistochemical analysis on HSERIV demonstrated that the marker pattern was similar to what is generally present in human skin in vivo. CONCLUSION HSERIV is morphologically and functionally compatible with human skin observed in vivo.

Assessment of inflammation based on the release of oxygen radicals by granulocytes in chronic uncontrolled asthma

OBJECTIVE To evaluate spontaneous release of superoxide anion by peripheral blood granulocytes of atopic patients with uncontrolled asthma undergoing glucocorticoid therapy and of healthy subjects. METHODS We studied 32 patients, aged 6 to 18 (mean 12.04), and 29 healthy subjects as a comparative group. Patients were grouped according to the forced expiratory vital capacity in the first second. Group I, forced expiratory vital capacity in the first second of between 60 and 80%, had 19 patients, and group II, forced expiratory vital capacity in the first second = 60%, had 13 patients. Spontaneous superoxide release by granulocytes was measured by a spectrophotometer method based on superoxide dismutase, before and after oral prednisone and beclomethasone, budesonide or fluticasone inhaled therapy. Statistical analyses were performed using ANOVA, Wilcoxon and Tukey tests. RESULTS Comparing the superoxide anion release by granulocytes of asthmatic patients and healthy subjects, we observed a higher release by cells of the uncontrolled patient group II (p < 0.05). Evaluating the superoxide release by cells of asthmatic patients before and after steroid therapy, a significant decrease was found only in patient group I. CONCLUSION The impact of corticosteroids on inflammatory modulation occurred in the uncontrolled asthmatics with forced expiratory vital capacity in the first second between 60 and 80%. In those with forced expiratory vital capacity in the first second of = 60%, this finding was not observed. Further studies are necessary to evaluate the effect of this finding on asthmatic patients.

Glucose-6-phosphate dehydrogenase deficiency with recurrent infections: case report

OBJETIVO: relatar a ocorrencia de uma deficiencia funcional de neutrofilos rara, com quadro clinico e laboratorial semelhante ao da doenca granulomatosa cronica. METODOS: relato de caso de paciente com deficiencia acentuada da glicose-6-fosfato desidrogenase e infeccoes de repeticao. Realizada pesquisa bibliografica utilizando as bases de dados Medline e Lilacs, abrangendo o periodo de 1972 a 2000. RESULTADOS: paciente com nivel da glicose-6-fosfato desidrogenase extremamente reduzido e quadro de infecoes graves com melhora clinica apos uso de cotrimoxazol continuo. Os leucocitos do paciente apresentam defeito no metabolismo oxidativo, similar ao da doenca granulomatosa cronica. CONCLUSOES: o diagnostico da deficiencia da glicose-6-fosfato desidrogenase em neutrofilos deve ser considerado em qualquer paciente com anemia hemolitica nao esferocitica congenita no qual o nivel da glicose-6-fosfato desidrogenase esteja anormalmente baixo ou apresente infecoes de repeticao. E diagnostico diferencial da doenca granulomatosa cronica.

Evaluation of Culture Medium for Human Keratinocytes

Human keratinocytes are needed for tissue engineering of skin applied in tissue repair and regeneration aimed at clinical application. The need to have high cell concentrations within a short time when performing cell proliferation is vital. The growth and maintenance of these cells commonly involve the use of MCDB 153 medium, which is supplemented with animal-derived components, such as growth factors and fetal bovine serum (FBS). The aim of this work was to evaluate different formulations based on MCDB 153 medium for keratinocyte proliferation. For that, several experiments were realized to define which supplements are important for skin cell culture using 24 factorial design. Skin samples were obtained from four consenting patients submitted to dermolipectomy, the plastic surgery operation. These fragments were treated with 0.25% trypsin-EDTA for four hours, at 37oC. Furthermore, the epidermis was separated from the dermis, providing skin cells. Batch cultures were performed in 6 and 96-well plates, in addition to 25 cm2 flasks. Results concerning cell growth and metabolism showed that keratinocytes were full grown up in MCDB 153 medium containing insulin (7.5 µg.mL-1), bovine pituitary extract-BPE (80 µg.mL-1), epidermal growth factor-EGF (0.08 µg.mL-1), hydrocortisone (0.63 µg.mL-1) and glutamine (1 g.L-1). On the other hand, culture media proposed in the experimental design did not resulted in satisfactory cell growth. In addition, although, the initial glucose concentration was low in the most of cases, the lactate was strongly produced by cells reaching 1 g.L-1.

Avaliação da inflamação com base na liberação de radicais oxidantes por granulócitos na asma crônica não-controlada

OBJECTIVE: To evaluate spontaneous release of superoxide anion by peripheral blood granulocytes of atopic patients with uncontrolled asthma undergoing glucocorticoid therapy and of healthy subjects. METHODS: We studied 32 patients, aged 6 to 18 (mean 12.04), and 29 healthy subjects as a comparative group. Patients were grouped according to the forced expiratory vital capacity in the first second. Group I, forced expiratory vital capacity in the first second of between 60 and 80%, had 19 patients, and group II, forced expiratory vital capacity in the first second = 60%, had 13 patients. Spontaneous superoxide release by granulocytes was measured by a spectrophotometer method based on superoxide dismutase, before and after oral prednisone and beclomethasone, budesonide or fluticasone inhaled therapy. Statistical analyses were performed using ANOVA, Wilcoxon and Tukey tests. RESULTS: Comparing the superoxide anion release by granulocytes of asthmatic patients and healthy subjects, we observed a higher release by cells of the uncontrolled patient group II (p < 0.05). Evaluating the superoxide release by cells of asthmatic patients before and after steroid therapy, a significant decrease was found only in patient group I. CONCLUSION: The impact of corticosteroids on inflammatory modulation occurred in the uncontrolled asthmatics with forced expiratory vital capacity in the first second between 60 and 80%. In those with forced expiratory vital capacity in the first second of = 60%, this finding was not observed. Further studies are necessary to evaluate the effect of this finding on asthmatic patients.