Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells.

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched.

Essential role of nuclear factor-kappaB for NADPH oxidase activity in normal and anhidrotic ectodermal dysplasia leukocytes.

This work investigated the functional role of nuclear factor-kappaB (NF-kappaB) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes CYBB, CYBA, NCF1, and NCF2. U937 cells with a stably transfected repressor of NF-kappaB (IkappaBalpha-S32A/S36A) demonstrated significantly lower superoxide release and lower CYBB and NCF1 gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), an inherited disorder of NF-kappaB function. Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91(0) CGD). NCF1 gene expression in EDA-ID S32I cells was decreased compared with healthy control cells and similar to that in autosomal recessive (A47(0)) CGD cells. Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-kappaB site 5' to the CYBB gene in U937 cells treated with NF-kappaB inhibitors, repressor-transfected U937 cells, and EDA-ID patients' cells. Zymosan phagocytosis was not affected by transfection of U937 cells with the NF-kappaB repressor. These studies show that NF-kappaB is necessary for CYBB and NCF1 gene expression and activation of the phagocyte NADPH oxidase in this model system.

A topical cell therapy approach for diabetic chronic ulcers: Effects of mesenchymal stromal cells associated with platelet-rich plasma.

BACKGROUND: Diabetic cutaneous ulcers are subjected to several physiological and biochemical defects, which contribute to wound chronicity and therapeutic failure. Platelet-rich plasma (PRP) has been used for stimulating tissue regeneration, and mesenchymal stromal cells (MSCs) have demonstrated therapeutic properties in all phases of skin regeneration in cell therapy studies. AIMS: The objective of this study was to evaluate the therapeutic effects related to the use of a biomembrane composed of autologous MSCs and PRP on chronic wounds of diabetic patients (pre-post pilot study). PATIENTS/METHODS: Six diabetic patients with chronic wounds for more than 6 months were subjected to adipose tissue collection for isolation of MSCs, blood collection for PRP preparation, and topical administration of a biomembrane of MSCs and PRP on each chronic wound. The statistical difference regarding the evolution of ulcers was calculated by means of paired t test. RESULTS: There was granulation tissue formation starting from 7 days after topical application. Total re-epithelialization occurred in 5 of the 9 lesions treated, and the mean wound healing rate (WHR) was 74.55% (±32.55%) after 90 days. No cicatricial hypertrophy or retraction was observed. CONCLUSION: Mesenchymal stromal cells topical therapy associated with PRP is well-tolerated and able to provide a reduction in ulcer area of diabetic chronic wounds.

Effects of BAY 41-2272, an activator of nitric oxide-independent site of soluble guanylate cyclase, on human NADPH oxidase system from THP-1 cells.

We investigated the effects of the 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b] pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) on the NADPH oxidase activity, gp91(phox) gene expression, cyclic guanosine-3',5'-monophosphate (cGMP) and cyclic adenosine-3',5'-monophosphate (cAMP) levels in the human myelomonocytic THP-1 cell line. THP-1 cells treated with BAY 41-2272 (0.3-10 microM) for 48 h significantly increased the superoxide anion (O(2)(*-)) release. This increase was not affected when cells were pre-treated with the specific cGMP-phosphodiesterase inhibitor zaprinast, the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxidiazolo[4,3-alpha] quinoxalin-1-one (ODQ), the adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl) adenine (SQ 22,536) or the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME). In addition, BAY 41-2272 (3 and 10 microM; 48 h) was able to increase gp91(phox) gene expression on THP-1 cells. The pre-treatment with zaprinast, 3-isobutyl-l-methyl-xanthine (IBMX; 0.5 mM), ODQ, SQ 22,536 or l-NAME caused no additional effect on the expression of gp91(phox) evoked by BAY 41-2272. Treatment of THP-1 cells with BAY 41-2272 caused a significant increase in cGMP and cAMP levels. Our findings show that BAY 41-2272 caused a significant increase on the O(2)(*-) release and gp91(phox) gene expression by THP-1 cells, and an elevation of intracellular cGMP and cAMP levels. However, we could not detect a clear correlation between both O(2)(*-) release and gp91(phox) gene expression with activation of cGMP and cAMP signaling pathways.

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.

The effect of IFN-gamma and TNF-alpha on the NADPH oxidase system of human colostrum macrophages, blood monocytes, and THP-1 cells.

The aim of this work was to analyze the effect of Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in human colostrum macrophages (CM), peripheral blood monocytes (PBM), and myelomonocytic THP-1 cells. We also investigated the effect of IFN-gamma on the release of TNF-alpha by these cells. Our results show that under basal culture conditions, CM release more superoxide than PBM and THP-1 cells (p < 0.05). The addition of IFN-gamma, alone or in combination with TNF-alpha, increased spontaneous superoxide release by PBM and THP-1 cells (p < 0.05) and increased phorbol myristate acetate (PMA)-stimulated superoxide release by CM, PBM, and THP-1 cells (p < 0.05). The NADPH oxidase activity of THP-1 cells consistently remained lower than that of CM or PBM, despite a dramatic response to IFN-gamma and TNF-alpha. Under basal conditions, gp91-phox gene expression was significantly higher in CM and PBM compared with THP-1 cells (p < 0.05). The addition of IFN-gamma alone or in combination with TNF-alpha caused a dramatic increase in gp91-phox gene expression in THP-1 cells (p < 0.05) but not in CM or PBM. Under basal conditions or in the presence of IFN-gamma, CM released more TNF-alpha than PBM or THP-1 cells (p < 0.05). In addition, PBM released more TNF-gamma than THP-1 cells (p < 0.05). IFN-gamma did not significantly augment the release of TNF-alpha by these cells (p > 0.05). Thus, IFN-gamma and TNF-alpha induced equivalent gp91-phox gene expression in THP-1 cells compared with CM or PBM but did not bring about equivalent NADPH oxidase activity. TNF-alpha release was higher in more mature cells. This partial divergence of gp91- phox gene expression, NADPH oxidase activity, and TNF-alpha release is probably a consequence of different events of myeloid cell biology and relates at least in part to cell differentiation state.

Melanocyte transplantation for the treatment of vitiligo: effects of different surgical techniques.

This paper presents the results of a pilot clinical trial study, conducted on 11 patients with stable vitiligo at the vitiligo outpatient clinic of The Unicamp University Hospital, between March 2000 and December 2001. This study was in accordance with the ethical standards of the Institutional Review Board. The patients were concomitantly treated with four different types of surgical techniques in 44 areas randomly chosen. There was a 90-day follow-up period. The following treatments were carried out: only cryotherapeutic treatment (OC); cryotherapy plus melanocyte culture medium (CM); cryotherapy plus transplantation of non-cultured melanocytes and keratinocytes (KM); and cryotherapy plus transplantation of cultured melanocytes (CC). The appearance of repigmentation and its evolution were followed all along the treatments. In the case of OC and CM no repigmentation occurred. Progressive repigmentation was observed over a period of 90 days in the case of KM and CC. In these two groups there was a significant reduction in the achromic areas during this time but no significant difference was found between the two treatments.

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.

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.

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, Masson's 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.

Up-regulation of NADPH oxidase components and increased production of interferon-gamma by leukocytes from sickle cell disease patients.

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P=0.036). Monocytes from SCD patients showed higher levels of p47(phox) phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P=0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P>0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Assessment of inflammation based on the release of oxygen radicals by granulocytes in chronic uncontrolled asthma / Avaliação da inflamação com base na liberação de radicais oxidantes por granulócitos na asma crônica não-controlada

Objetivo: Avaliar a liberação espontânea de ânion superóxidopor granulócitos de sangue periférico de pacientescomasma crônica não-controlada antes e após corticoterapia e de indivíduos sadios. Métodos: Foram estudados 32 pacientes entre 6 e 18 anos (média 12,04 anos) e 29 indivíduos sadios como grupo decomparação.Os pacientes oramagrupados de acordocomo volumeexpiratório forçado no primeiro segundo: grupo I, volume expiratórioforçado no primeiro segundo entre 60 e 80%, 19 pacientes; e grupo II, volume expiratório forçado no primeiro segundo = 60%, 13 pacientes. A liberação espontânea de superóxido por granulócitos, medida por espectrofotometria utilizando superóxido dismutase, foi avaliada nos pacientes antes e após o tratamento com prednisona por via oral e beclometasona, budesonida ou fluticasona administradas por via inalatória. Na análise estatística foramutilizados os testes de análise de variância, Tukey e de Wilcoxon.Resultados: Comparando-se a liberação de ânion superóxido por granulócitos dos pacientes asmáticos e indivíduos sadios observamosque a liberação foi maior nos asmáticos não-controladosdo grupo II (p < 0,05). Avaliando-se a liberação de superóxido pelas células dos pacientes antes e após a terapiacomcorticosteroideuma diminuição significativa foi observada apenas no grupo I. Conclusão: O impacto dos glicocorticoides sobre a modulação da inflamação ocorreu nos indivíduos asmáticos não-controlados com volume expiratório forçado no primeiro segundo entre 60 e 80%.Naqueles com volume expiratório forçado no primeiro segundo = 60não foi observada essa modulação, havendo necessidade de mais estudos para avaliar o impacto de tal achado nos pacientes asmáticos.

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 weregrouped according to the forced expiratory vital capacity in the firstsecond. Group I, forced expiratory vital capacity in the first second of between60and80%,had 19 patients, and group II, forced expiratoryvital capacity in the first second=60%,had 13 patients. Spontaneous superoxide release by granulocytes was measured by aspectrophotometer 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 bygranulocytes of asthmatic patients and healthy subjects,weobserved 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 inflammatorymodulation 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 findingwas not observed. Further studies are necessary to evaluate the effect of this finding on asthmatic patients.

Immunoarchitectural characterization of a human skin model reconstructed in vitro / Caracterização morfológica e imunoistoquímica de um modelo de pele humana reconstruída 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, Masson's 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 (34βE12), low molecular weight cytokeratins (35βH11), 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.

CONTEXTO E OBJETIVO: Nos últimos anos, diferentes modelos de pele humana reconstruída in vitro (PHRIV) foram descritos para uso clínico e aplicações em pesquisa na indústria farmacêutica. Antes de serem liberados para uso rotineiro como substitutos de pele humana, os modelos de PHRIV necessitam de testes (estudos) comparativos com a pele humana in vivo, por meio de análises morfológica (arquitetural) e imunoistoquímica (funcional). O objetivo deste trabalho é estudar as características imunoistoquímicas de um modelo de PHRIV desenvolvido em nosso serviço, comparando-as com a pele humana, para que esse modelo de PHRIV possa vir a ser testado clinicamente em casos de queimaduras e ulcerações de pele nos quais métodos tradicionais de tratamento não estejam indicados. TIPO DE ESTUDO E LOCAL: Estudo experimental laboratorial realizado no Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas (FCM/Unicamp), Campinas, São Paulo, Brasil. MÉTODOS: Cortes histológicos foram corados com hematoxilina-eosina, tricrômio de Masson para fibras colágenas, ácido periódico-reagente de Schiff para membrana basal e glicogênio, Weigert-Van Gieson para fibras elásticas e Fontana-Masson para melanócitos. Estudo imunoistoquímico foi realizado para identificar citoqueratinas de amplo espectro de pesos moleculares (AE1/AE3), citoqueratinas de alto peso molecular (34βE12), citoqueratinas de baixo peso molecular (35βH11), citoqueratinas 7 e 20, vimentina, proteína S-100 (para melanócitos e células dendríticas), CD68 (KP1, histiócitos) e CD34 (QBend, endotélio). RESULTADOS: A histologia revelou similaridade satisfatória entre PHRIV e a pele in vivo. O estudo imunoistoquímico da PHRIV demonstrou padrão semelhante de marcadores usualmente presentes na pele humana in vivo. CONCLUSÃO: A PHRIV estudada é morfológica e funcionalmente compatível com a pele humana observada in vivo.

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

CONTEXTO E OBJETIVO: A técnica para obtenção de pele humana que apresente derme e epiderme, reconstruída a partir de células isoladas de pacientes, pode possibilitar a realização de enxertos autólogos de pele reconstruída em laboratório em pacientes com áreas doadoras escassas, além de permitir ensaios com substâncias químicas e drogas in vitro e não mais in vivo. O objetivo do trabalho é demonstrar um método de obtenção de pele humana reconstruída in vitro composta de derme e epiderme associadas. TIPO DE ESTUDO E LOCAL: Estudo experimental laboratorial realizado no Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas, Campinas, São Paulo, Brasil. MÉTODOS: A partir da cultura de fibroblastos humanos, é possível obter um número suficiente de células que podem ser injetadas em uma matriz de colágeno bovino tipo I que, mantida imersa em meio de cultura específico para fibroblastos, permite a formação de uma derme humana reconstruída in vitro. Sobre essa derme, por meio de cultura de queratinócitos e melanócitos humanos, forma-se epiderme diferenciada, levando à formação de pele humana reconstruída in vitro, composta de derme e epiderme associadas. RESULTADOS: Demonstramos que é possível reproduzir pele humana reconstruída in vitro, composta de derme e epiderme associadas. Essa pele humana formada é, histologicamente, semelhante à pele humana in vivo. Na derme, identifica-se o tecido colágeno, com suas células, e a matriz extracelular organizados paralelamente à epiderme. Esta se desenvolve em várias camadas. CONCLUSÃO: É possível obter pele humana reconstruída in vitro, completamente diferenciada, composta de derme e epiderme, associadas, a partir da injeção de fibroblastos humanos em uma matriz de colágeno bovino tipo I e da cultura seqüencial de queratinócitos e melanócitose humanos sobre essa matriz contendo fibroblastos em seu interior.

Atividade da NADPH oxidase em granulócitos e células mononucleares de adolescentes e crianças asmáticos segundo a gravidade da doença / NADPH oxidase activity of granulocytes and mononuclear cells of asthmatic children according to the disease severity

Objetivo: Investigar a liberaçäo espontânea e estimulada (com forbol miristato acetato - PMA) de superóxido por granulócitos e células mononucleares de adolescentes e crianças asmáticos classificadas segundo os critérios do relatório Iniciativa Global para Asma (GINA) de 1997. Métodos: Selecionamos 30 pacientes de seis a quinze anos, e os classificamos como tendo asma intermitente leve (AIL, n=9), persistente leve (APL,n=8), persistente moderada (APM,n=7) e persistente grave (APG, n=6). Os granulócitos e células mononucleares foram fracionados a partir de amostras do sangue periféricos por gradiente de densidade descontínuo. A cinética de liberaçäo de superóxido (0,5,15,25, 45, e 60 minutos) foi avaliada segundo a reduçäo do citocromo c, especificamente inibida pela superóxido dismutase. Os resultados foram comparados com os de 18 adultos sadios por análise de variância. Resultados: A liberaçäo espontânea de superóxido pelos granulócitos foi significamente maior, aos 25 minutos, nos grupos APL, APM e APG comparado aos grupos de indivíduos sadios e AIL (p<0,05), aos 45 minutos nos grupos APL e APM e aos 60 minutos nos grupos APG. A liberaçäo de superóxido pelos granulócitos estimulados com PMA, foi significamente maior nos grupos APM e APG que nos indivíduos sadios, em todos os tempos. Näo houve diferença significativa na liberaçäo de superóxido por células mononucleares, estimuladas ou näo com PMA, entre os diversos grupos de asmáticos e indivíduos sadios

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

CONTEXTO: Recentes progressos no campo das técnicas de cultura epitelial têm levado ao desenvolvimento de sistemas de cultura nos quais a epiderme reconstruída obtida exibe características de diferenciação morfológica semelhantes àquelas vistas in vivo. Uma epiderme humana reconstruída in vitro pode ser utilizada como melhor alternativa para testes toxicológicos e de eficácia de produtos de uso tópico in vitro e ainda no tratamento de queimaduras e úlceras crônicas de pele. OBJETIVO: Demonstrar um método de obtenção de epiderme humana reconstruída in vitro, utilizando queratinócitos e melanócitos cultivados sobre uma derme humana morta desepidermizada. TIPO DE ESTUDO: Experimental Laboratorial. LOCAL: Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas, Campinas, São Paulo, Brasil. PROCEDIMENTOS: Queratinócitos e melanócitos humanos cultivados in vitro foram semeados sobre uma matriz biológica (derme humana morta desepidermizada) e o sistema foi mantido em interface ar-líquido, em meio de cultura adequado, até haver a formação de uma epiderme humana estratificada, mantendo as características histológicas da epiderme in vivo. RESULTADOS: Demonstramos, histologicamente, que é possível reproduzir uma epiderme diferenciada, a partir da cultura de queratinócitos e melanócitos sobre uma derme humana morta desepidermizada, obtendo uma epiderme humana reconstruída in vitro, com queratinócitos e melanócitos funcionais, corretamente posicionados, equivalente à epiderme in vivo. CONCLUSÕES: É possível obter uma epiderme humana reconstruída in vitro completamente diferenciada a partir da cultura de queratinócitos e melanócitos sobre uma derme humana morta desepidermizada.

Papel do fator nuclear kappa B (NF-κB) na expressâo do gene NCF1 em leucócitos de indivíduos normais, e pacientes com doença granulomatosa crônica, displasia ectodérmica anidrótica, ou com defeitos no eixo IL-12/23-IFN-γ / Role of nuclear factor kappa B (NF-κB) on NCF1 gene expression in leukocytes from normal individuals, and patients with chronic granulomatous disease, anhidrotic ectodermal dysplasia, or with defects in the IL-12/23/IFN-γ axis

Objetivo: Analisamos a relevância do NF-κB sobre a expressão do gene NCF1 em células mielóides U937 selvagens (U937) ou transfectadas com um repressor do NF-κB (IκBα-S32A/S36A - U937 IκBα-S32A/S36A) ou transfectadas com o vetor vazio (U937 pCMV3) e em células B imortalizadas pelo vírus Epstein-Barr (EBV) de pacientes com displasia ectodérmica anidrótica com imunodeficiência (EDA-ID) ou com doença granulomatosa crônica (CGD) devido a mutações no gene NCF1, ou de pacientes portadores de defeitos do eixo IL-12/ 23-IFN-γ. Métodos: O RNA celular total foi isolado pelo método TRI-zol®. Os cDNAs foram produzidos utilizando-se o SuperScript™ III e amplificados por Real-time PCR (SYBR® Green Master Mix). Resultados: Células U937 IKBα-S32A/S36A mostraram significante decréscimo na expressão do gene NCF1 comparadas com as células U937. A expressão do gene NCF1 em células EDA-ID S32I foi significativamente menor que em controles saudáveis, assim como em células EDA-ID NEMO/IKKγ X420W na mesma comparação. Estes resultados foram similares aos encontrados em células de pacientes CGD devido à mutação autossômica recessiva no gene NCF1 quando comparados com o controle normal. Defeitos nos receptores IFNGR1 e IFNGR2 levam à diminuição da expressão do gene NCF1 (p<0,05, Mann Whitney). Conclusões: Estes resultados mostram que o NF-κB é necessário para a expressão do gene NCF1, que possivelmente as subunidades p50 e/ou p65 do NF-κB ligam-se funcionalmente à região "upstream" do gene NCF1 e que defeitos no eixo IL-12/ 23-IFN-γ influenciam a expressão do gene NCF1.

Objective: We analyzed the relevance of NF-κB on NCF1 gene expression in regular myeloid U937 cells (U937), or transfected with a NF-κB repressor (IκBo-S32A/S36A - U937 IκBo-S32A/S36A), or transfected with the empty vector (U937 pCMV3), and in B cells immortalized by Epstein-Barr vírus (EBV) from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), or with chronic granulomatous desease (CGD) due to mutations in NCF1 gene, or from patients with IL-12/23-IFN-γ axis defects. Methods: Total RNA was isolated by the TRIzol® method. The cDNAs were produced by Super Script™ III and amplified by Real-time PCR (SYBR Green Master Mix). Results: U937 IκBo-S32A/S36A cells showed significant decrease in the NCF1 gene expression compared with the U937 cells. The NCF1 gene expression was significantly lower in EDA-ID S32I cells compared to healthy controls as well as in EDA-ID NEMO/IKKy X420W cells. These results were similar to those obtained in the CGD patient cells due recessive mutations in the NCF1 gene compared to healthy controls. Cells form patients with defects in IFNGR1 e IFNGR2 receptors also presented decreased NCF1 gene expression (p<0,05, Mann Whitney). Conclusion: These results show that the NF-κB is necessary for NCF1 expression, possibly the p50 and/or p65 NF-κB subunits bind functionally to the upstream region of the NCF1 gene and that IL-12/23-IFN-γ axis defects also influence NCF1 gene expression.

Deficiência da glicose-6-fosfato desidrogenase com infeções de repetição: relato de caso / Glucose-6-phosphate dehydrogenase deficiency with recurrent infections: case report

Objetivo: relatar a ocorrência de uma deficiência funcional de neutrófilos rara, com quadro clínico e laboratorial semelhante ao da doença granulomatosa crônica. Métodos: relato de caso de paciente com deficiência acentuada da glicose-6-fosfato desidrogenase e infecções de repetição. Realizada pesquisa bibliográfica utilizando as bases de dados Medline e Lilacs abrangendo o período de 1972 a 2000. Resultados: paciente com nível da glicose-6-fosfato desidrogenase extremamente reduzido e quadro de infecções graves com melhora clínica após uso de cotrimoxazol contínuo. Os leucócitos do paciente apresentam defeito no metabolismo oxidativo, similar ao da doença granulomatosa crônica. Conclusões: o diagnóstico da deficiência da glicose-6-fosfato desidrogenase em neutrófilos deve ser considerado em qualquer paciente com anemia hemolítica não esferocítica congênita no qual o nível da glicose-6-fosfato desidrogenase esteja anormalmente baixo ou apresente infecções de repetição. É diagnóstico diferencial da doença granulomatosa crônica