Histology and histopathology Vol.26, nº6 (2011)
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- PublicationOpen AccessExcavation of a buried treasure – DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Klopfleisch, R.; Weiss, A.T.A; Gruber, A.D.Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.
- PublicationOpen AccessExpression of cell-cycle regulatory proteins BUBR1, MAD2, Aurora A, cyclin A and cyclin E in invasive ductal breast carcinomas(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Du, Juan; Du, Qiang; Zhang, Yang; Sajdik, Constantin; Ruan, Yuan; Tian, Xin-xia; Fang, Wei-gangCyclin A, cyclin E, BUBR1, MAD2 and Aurora A are all cell-cycle regulatory proteins and have been proven to play crucial roles in carcinogenesis. However, their expression patterns in invasive ductal breast carcinoma (IDBC) are controversial and unclear. In this study, we examined the expression status of these candidate proteins in a set of 117 invasive ductal carcinomas, and evaluated their associations with known clinicopathological parameters and the expressions of estrogen receptor, progesterone receptor, Ki-67 and Her-2. Univariate and multivariate data analyses both displayed that positive BUBR1 expression was associated with a high Ki-67 labeling index, and negative MAD2 expression was associated with Her-2 overexpression. Positive BUBR1 expression was also associated with a high histological tumor grade in univariate analysis, but not in multivariate analysis. In addition, high Aurora A expression was weakly associated with lymph node metastasis, and cyclin A was strongly associated with the expression of cyclin E in both univariate and multivariate models. In conclusion, this study suggests that evaluation of BUBR1, MAD2 and Aurora A expression levels is likely to improve accuracy of prognostic predictions in IDBC.
- PublicationOpen AccessVisualisation and stereological assessment of blood and lymphatic vessels(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Lokmic, Zerina; Mitchell, Geraldine M.The physiological processes involved in tissue development and regeneration also include the parallel formation of blood and lymphatic vessel circulations which involves their growth, maturation and remodelling. Both vascular systems are also frequently involved in the development and progression of pathological conditions in tissues and organs. The blood vascular system circulates oxygenated blood and nutrients at appropriate physiological levels for tissue survival, and efficiently removes all waste products including carbon dioxide. This continuous network consists of the heart, aorta, arteries, arterioles, capillaries, post-capillary venules, venules, veins and vena cava. This system exists in an interstitial environment together with the lymphatic vascular system, including lymph nodes, which aids maintenance of body fluid balance and immune surveillance. To understand the process of vascular development, vascular network stability, remodelling and/or regression in any research model under any experimental conditions, it is necessary to clearly and unequivocally identify and quantify all elements of the vascular network. By utilising stereological methods in combination with cellular markers for different vascular cell components, it is possible to estimate parameters such as surface density and surface area of blood vessels, length density and length of blood vessels as well as absolute vascular volume. This review examines the current strategies used to visualise blood vessels and lymphatic vessels in two- and three-dimensions and the basic principles of vascular stereology used to quantify vascular network parameters.
- PublicationOpen AccessCalcium, calcium-sensing receptor and growth control in the colonic mucosa(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Varani, JamesA role for calcium in epithelial growth control is well-established in the colon and other tissues. In the colon, Ca2+ “drives” the differentiation process. This results in sequestration of ß-catenin in the cell surface / cytoskeletal complex, leaving ß-catenin unavailable to serve as a growth-promoting transcription enhancer in the nucleus. The signaling events that lead from Ca2+ stimulation to differentiation are not fully understood. A critical role for the extracellular calcium-sensing receptor (CaSR) is assumed, based on CaSR localization to the differentiating epithelial cells in the normal colonic mucosa (upper half of the crypt and crypt surface), decreased CaSR expression in colon carcinoma, and the results from in vitro studies with colonic epithelial cell lines. While Ca2+ is well-accepted as a growth-regulating agent in the colon, suppression of cell proliferation is not complete. At least part of the reason for this is the inherent variability in Ca2+ responsiveness among individual epithelial cells. Of interest, colon epithelial cells that are resistant to the growth-regulating activity of Ca2+ alone are still responsive to Ca2+ in conjunction with other transition metals. Whether a multi-mineral approach will, ultimately, prove to be more effective than Ca2+ alone as a colon cancer chemopreventive agent remains to be seen, but certainly worth investigating.
- PublicationOpen AccessInduction of epithelial migration of lymphocytes by intercellular adhesion molecule-1 in a rat model of oral mucosal graft-versus-host disease(Editores F. Hernandez y Juan F. Madrid. Murcia, Universidad de Murcia, Departamento de Biologia Celular e Histologiam, 2011) Ohno, Jun; Iwahashi, Teruaki; Ehara, Michiko; Ozasa, Ryuki; Hanada, Hironori; Funakoshi, Tomoyuki; Taniguchi, KunihisaTo elucidate the involvement of intercellular adhesion molecule-1 (ICAM-1) in the migration of lymphocytes to the oral mucosal epithelium in a rat model of acute graft-versus-host disease (AGVHD), we investigated (1) ICAM-1 and major histocompatibility complex (MHC) class II expression by keratinocytes (KCs) and their role in the epithelial infiltration of CD8+ cells, (2) the tissue expression of interferon-γ (IFN-γ) mRNA and expression of IFN-γ receptor by KCs, and (3) the ability of KCs to direct CD8+ cells into the epithelial layers. We classified the oral mucosal lesions into three consecutive temporal phases on the basis of increased epithelial ICAM-1expression: basal- (phase I), parabasal- (phase II), and pan-epithelial except for the cornified cell layer (phase III). Basal ICAM-1 expression by KCs preceded that of MHC class II molecules, infiltration of CD8+ cells and epithelial histological changes. Tissue expression of IFN-γ mRNA and expression of IFN-γ receptor on KCs evidenced by immunohistochemistry were detected in early lesions (phase I), indicating that locally produced IFN-γ induced ICAM-1 expression by KCs. CD8+ cells were bound to KCs in frozen sections of epithelial lesions, whereas no lymphocyte attachment was observed in normal KC. Adherence could be inhibited by pretreating CD8+ cells with lymphocyte function-associated antigen-1 (LFA-1) antibody and/or by pretreating sections with ICAM-1 antibody. Our data suggest that in the early phase of acute oral mucosal GVHD, the induction of ICAM-1 expression on KCs leads to the migration of CD8+ cells into the epithelium and that this is mediated in part by the ICAM-1/LFA-1 pathway.
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