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 (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. 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Marcadores microssatélites relacionados com a resistência à vassoura-de-bruxa do cacaueiro Microsatellite markers related to resistance of cocoa tree against witches'-broom

Os objetivos deste trabalho foram caracterizar a resistência à vassoura-de-bruxa de plantas de cacau originadas do cruzamento entre TSH 1188 e CCN 51 (população segregante), por meio de dois métodos de inoculação em condições de campo, e identificar marcadores microssatélites específicos para grupos de plantas resistentes e suscetíveis. As plantas-controle avaliadas pelos métodos de inoculação natural e inoculação artificial em campo produziram os mesmos padrões de sintomas. As plantas da população segregante também coincidiram os padrões de sintomas em 90%, por esses dois métodos. O método de inoculação artificial em campo permite detectar falso-resistentes. Dos 18 pares de primers microssatélites amplificados, 15 foram polimórficos entre os genitores, e seis entre os grupos de plantas segregantes contrastantes quanto à resistência à vassoura-de-bruxa. Foram confirmadas três marcas previamente associadas a QTL (locos para características quantitativas) relacionados com a resistência à vassoura-de-bruxa, comuns a outras populações. Também foram identificados três novos QTL para esta característica, típicos desta população, o que comprova sua utilidade para o melhoramento genético do cacaueiro.<br>The objectives of this work were to evaluate cocoa tree resistance against witches'-broom, in plants originated from the crossing between TSH 1188 and CCN 51 (segregating population), by means of two methods of inoculation in field conditions, and to identify microsatellite markers specific for resistant and susceptible plants. The control plants bore identical symptoms as the plants of the segregating population in 90% of the cases under the two methods. The method of artificial inoculation in the field allows the detection of false resistance to the disease. Of the 18 pairs of microsatellite primers amplified, 15 were polymorphic between genitors and six were polymorphic between the two groups of plants evaluated for resistance to witches'-broom. Three previously characterized markers were confirmed as associated to QTL (quantitative trait loci) related to resistance to the witches'-broom, which is common in other populations. Three new QTL for this characteristic, typical of this population, were also identified, which proves the utility of this population for cocoa breeding

Diversidade genética entre acessos de cacau de fazendas e de banco de germoplasma na Bahia Genetic diversity among farm and germplasm bank accessions of cacao in Bahia, Brazil

O objetivo deste trabalho foi avaliar a diversidade genética de acessos de cacau, selecionados previamente como produtivos e resistentes à vassoura-de-bruxa na Bahia, e estudar suas inter-relações com genótipos no banco de germoplasma. Amostras de DNA de folhas dos 120 acessos, coletados em 17 fazendas de sete municípios do Sul da Bahia, foram amplificadas pela técnica de RAPD ("random amplified polymorphic DNA"). Os coeficientes de dissimilaridade genética, calculados pelo método de Jaccard a partir das bandas RAPD, permitiram evidenciar, pela análise de agrupamento, que a maioria das seleções das fazendas (89,2%) agrupou-se com acessos do banco de germoplasma considerados representativos da diversidade de cacau (híbridos, trinitários, Scavinas, amazônicos e cacau-comum). As demais seleções distribuíram-se em outros sete grupos distintos. Há elevada diversidade genética entre as seleções das fazendas, e algumas delas devem ter-se originado de genitores não incluídos nesta análise. Esses materiais apresentam potencial para seleção de clones com maior diversidade para novos cruzamentos ou uso pelos agricultores.<br>The objective of this work was to assess genetic diversity of farm cacao accessions, previously selected as productive and resistant to witch broom in Bahia, Brazil, and to study their interrelationship with reference genotypes on germplasm bank. DNA samples from 120 cacao accessions, collected on 17 farms in seven counties in Southern Bahia, were amplified by RAPD technique (random amplified polymorphic DNA). The coefficients of genetic dissimilarity, calculated from the RAPD bands by Jaccard method, allowed to show by cluster analysis that most farm selections (89.2%) grouped with accessions from germplasm bank considered representatives of the cacao genetic diversity (hybrids, Trinitarian, Scavina, Amazon and common cacao). The other selections were distributed in seven distinct groups. There is high genetic diversity among farm selections, and some of them may have originated from other progenitors not included in this analysis. These materials present potential for clone selection with higher diversity for new crossings or direct use by agriculturists

Sources of resistance to Crinipellis perniciosa in progenies of cacao accessions collected in the Brazilian Amazon Fontes de resitência a Crinipellis perniciosa em progênies de cacaueiros coletados na Amazônia brasileira

The witches' broom disease caused by the fungus Crinipellis perniciosa is the main phytossanitary constraint for cacao production in Brazil. The integrated management of the disease involves resistance as one of the components. The breeding program conducted by the Brazilian Institution, CEPLAC is directed toward the pyramidation of resistance genes from different sources to achieve a more durable resistance. This study aimed to identify sources of resistance in progenies of cacao accessions collected in the basins of ten Amazonian rivers and compared to progenies from the Peruvian clones 'Scavina 6' and 'Sacavina 12'. Progenies from 40 Amazonian accessions and 'Scavina' were evaluated in the field for six years for witches' broom resistance through multivariate and repeated measurement analyses evaluating the effect of progeny, area, block, year, and their interactions. There were differences in the mean number of vegetative brooms on some Amazonian progenies and 'Scavina' descendants. There was an increase in the number of vegetative brooms in the last year for 'Scavina' progenies, but that was not observed for the Amazonian progenies 64, 66, 156, 194, 195, 269 and 274. There were different gene/alleles for resistance in the Amazonian progenies in comparison to the traditional 'Scavina' accessions. These new sources of resistance will be important for pyramiding resistance genes and consequently increasing the stability and durability of the resistance to witches' broom.<br>A doença vassoura-de-bruxa, causada pelo fungo Crinipellis perniciosa, é o principal problema fitossanitário para o cultivo do cacaueiro no Brasil. O manejo integrado da doença envolve a resistência como um dos componentes. O programa de melhoramento genético do cacaueiro conduzido pela Instituição brasileira CEPLAC é direcionado para acumular genes de resistência de diferentes fontes visando à obtenção de uma resistência mais durável. O objetivo deste estudo foi identificar novas fontes de resistência em progênies de acessos de cacau da Amazônia brasileira. Os acessos foram coletados em dez bacias hidrográficas da Amazônia e comparados com progênies de 'Scavina 6' e 'Scavina 12', do Peru. Progênies de 40 acessos amazônicos e de 'Scavina' foram avaliadas no campo por seis anos para resistência à vassoura-de-bruxa, através de análise multivariada e de medidas repetidas considerando os efeitos de progênie, ensaio, bloco, ano e suas interações. Houve diferenças no número de vassouras vegetativas em algumas das progênies amazônicas e de 'Scavina'. Houve também incremento no número de vassouras para os descendentes de 'Scavina' no último ano do experimento, fato não observado para as progênies dos clones amazônicos 64, 66, 156, 194, 195, 269 e 274. Existem diferentes genes/alelos de resistência em progênies amazônicas e em descendentes do tradicional 'Scavina'. As novas fontes de resistência serão importantes para a piramidação de genes/alelos para a ampliação da estabilidade e durabilidade da resistência