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1.
Curr Protoc Essent Lab Tech ; 21(1): e46, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33381282

RESUMO

Stored biological materials should have minimal pre-analytical variations in order to provide researchers with high-quality samples that will give reliable and reproducible results, yet methods of storage should be easy to implement, with minimal cost and health hazard. Frozen tissue samples are a valuable biological resource. Here we compare different methods, such as liquid nitrogen (LN) or dry ice (DI), to a cheap and safe alternative using an aluminum platform (AP). Murine fresh liver and pancreas tissues were used with varying lengths of warm ischemia time. Quality assessment was based on histological evaluation, DNA and RNA extraction and quantification, and RNA degradation analysis, as well preservation of antigens for immunofluorescence, in a blinded manner. Both in superficial and deep tissue sections, based on histological assessment, AP is superior to DI, or as good as LN techniques in terms of presence of ice crystals, cutting artifacts, and overall quality/structural preservation. DNA and RNA were successfully extracted in reasonable quantities from all freezing techniques, but RNA degradation was seen for pancreas samples across all techniques. Immunofluorescence with cytokeratin8 (CK-8), alpha smooth muscle actin (αSMA), CD3, and B220 shows equally good outcomes for AP and LN, which are better than DI. The aluminum platform is a cheap, yet reliable method to freeze samples, rapidly preserving histological, antigenic, and DNA/RNA quality. Wider testing is required across different sample types. © 2020 The Authors. Basic Protocol: Flash-freezing fresh tissue with aluminum platform Alternate Protocol 1: Freezing fresh tissue with liquid nitrogen Alternate Protocol 2: Freezing fresh tissue with dry ice.

2.
Ann Diagn Pathol ; 47: 151557, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32593808

RESUMO

Recently, stromal targeting, by agents such as All trans retinoic acid (ATRA), has been regarded as a promising avenue for the treatment of pancreatic ductal adenocarcinoma (PDAC). The intra-cellular transportation of ATRA to the nuclear receptors is performed by either: fatty acid binding protein 5 (FABP5) or cellular retinoic acid binding protein 2 (CRABP2), dictating the transcription of downstream genes and, thus, eventual cell phenotype. Here, we explored the levels of each protein, in pancreatic tissues of patients presenting with a range of pancreatic diseases (pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), cholangiocarcinoma (CC)). We demonstrate that there is a significantly lower CRABP2 and FABP5 expression in activated fibroblasts or pancreatic stellate cells (PSC) in PDAC, as well as other diseased pancreas as in CC and CP, versus quiescent fibroblasts. The quiescent fibroblasts consistently show a pattern of high FABP5:CRABP2 ratio, whereas PSC in all non-PDAC tissues showed a low FABP5:CRABP2 ratio. PSC in PDAC patients had a range of FABP5:CRABP2 ratios (high, even and low). There was a lower CRABP2 expression in cancerous epithelial cells (PDAC) versus normal epithelial cells. This is also present in other disease states (CP, CC). Contrasting to the patterns seen for fibroblasts, the FABP5 expression in PDAC epithelial cells matched that of the normal epithelial cells. However, the normal epithelial cells had a high FABP5:CRABP2 ratio, compared to the PDAC epithelial cells. These ratios may have correlation with tumor progression, and overall survival. These findings could be confirmed in in vitro cell lysates. CRABP2 and FABP5 levels and ratios could serve as valuable biomarkers.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Pâncreas/patologia , Receptores do Ácido Retinoico/genética , Tretinoína/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Progressão da Doença , Células Epiteliais/metabolismo , Proteínas de Ligação a Ácido Graxo/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pâncreas/fisiopatologia , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Receptores do Ácido Retinoico/efeitos dos fármacos , Análise de Sobrevida , Análise Serial de Tecidos/métodos , Tretinoína/farmacologia , Tretinoína/uso terapêutico
3.
Gastroenterology ; 132(2): 527-30, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17258725

RESUMO

BACKGROUND & AIMS: We report a patient of Indian descent with parental consanguinity, who developed 10 carcinomas and 35 adenomatous polyps at age 23 and duodenal adenocarcinoma at age 25. He also had dysmorphic features, mental retardation, and café-au-lait spots but no brain tumor. We aimed to establish his molecular diagnosis. METHODS: Germ-line screening for APC and MYH/MUTYH mutations was normal as was immunohistochemistry for MLH1 and MSH2 proteins. Investigation by array-comparative genomic hybridization revealed deletion of a small region on chromosome 7. Using polymerase chain reaction, this region was refined to a 400-kilobase deletion, which included exons 9-15 of the PMS2 gene, and all coding regions of oncomodulin, TRIAD3, and FSCN1. RESULTS: The deletion was confirmed as homozygous, and both parents were carriers. Immunohistochemistry showed absent PMS2 expression in all tumors and normal tissue. Most tumors showed microsatellite instability, more marked at dinucleotide than mononucleotide repeats. The tumors harbored no somatic mutations in APC, BRAF, AXIN2, or beta-catenin, but KRAS2 and TGFBR2 mutations were found. CONCLUSIONS: Our patient represents a novel phenotype for homozygous PMS2 mutation and perhaps the most severe colorectal cancer phenotype-in terms of numbers of malignancies at an early age-described to date. PMS2 mutations-and perhaps other homozygous mismatch repair mutations-should be considered in any patient presenting with multiple gastrointestinal tumors, since our patient could not be distinguished clinically from cases with attenuated familial adenomatous polyposis or MUTYH-associated polyposis.


Assuntos
Pólipos Adenomatosos/genética , Adenosina Trifosfatases/genética , Neoplasias Colorretais/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Homozigoto , Polipose Intestinal/genética , Polipose Adenomatosa do Colo/diagnóstico , Pólipos Adenomatosos/diagnóstico , Pólipos Adenomatosos/patologia , Adulto , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , DNA Glicosilases/genética , Diagnóstico Diferencial , Neoplasias Duodenais/genética , Evolução Fatal , Regulação Neoplásica da Expressão Gênica , Humanos , Polipose Intestinal/diagnóstico , Polipose Intestinal/patologia , Masculino , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento , Mutação , Linhagem , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Índice de Gravidade de Doença , Proteínas ras
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