Histology and histopathology Vol.26, nº6 (2011)
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- 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 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 AccessRole of cannabinoid receptors and RAGE in inflammatory bowel disease(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Stintzing, Sebastian; Wissniowski, Till Th.; Lohwasser, Christina; Alinger, Beate; Neureiter, Daniel; Ocker, MatthiasBackground: The endocanabinoid system is involved in many inflammatory diseases, such as Crohn’s disease (CD) and ulcerative colitis (UC). The distribution and expression of cannabinoid receptors 1 (CNR1) and 2 (CNR2) in combination with inflammatory cytokines and RAGE (receptor of advanced glycation end products), which is also overactive in these diseases, in dependency of the extent of inflammation and alteration of the colon barrier is still unclear and needs to be elucidated. Material and Methods: 10 specimens of CD patients who underwent colectomy and 14 colectomy specimens of patients suffering from UC were investigated histologically for inflammatory infiltrate, extent of fibrosis and for disturbance of the intestinal barrier. Immunohistochemistry was carried out to examine the distribution and localization of CNR1, CNR2 and RAGE. Additionally, qRT-PCR was performed to study the expression of CNR1, CNR2, RAGE and inflammatory cytokines (TNFα, TGFß, CTGF, IL12, IFNγ). 35 morphological and histological normal specimens of colectomy cases served as controls. Results: The expression level of CNR2 did not differ between the control group and the group of patients with IBD, while CNR1 displayed a significant up regulation, especially in cases of CD. A differential association between the expression of CNR1/CNR2 and RAGE with morphological changes and expression of molecular markers of inflammation could be established. Conclusion: We showed that cannabinoid receptors are expressed differentially in inflammatory bowel disease and that the expression seems to be influenced by the underlying disease and by localized inflammation.
- PublicationOpen AccessEfficacy of Nigella sativa in alleviating benzo[a]pyrene-induced immunotoxicity in broilers(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Latif, I.K.; Karim, A.J.; Zuki, A.B.Z.; Zamri-Saad, M.; Niu, J.P.; Noordin, M.M.The immune response of broiler chickens exposed to intra-tracheal (i.t.) administration of benzo[a]pyrene (BaP) with and without Nigella sativa (Ns) supplementation was investigated. A total of 120 day-old chicks were divided into four groups comprising 30 birds each, into a control, Ns, BaP, and BaP+Ns group. Immune responses to Newcastle disease (ND) were evaluated by haemagglutination inhibition (HI), phytohaemagglutinin (PHA) skin test and carbon clearance assay (CCA). In most instances, there was a significant increase (p<0.05) in the ND-HI antibody titers, PHA skin-swelling response and phagocytic activity in the BaP + Ns group compared to that of the BaP group. Likewise, organ weight and indices of the spleen, bursa of Fabricius and thymus of birds from the BaP + Ns group were also higher (p<0.05) than that of the BaP group from day 1 until day 21. It is concluded that exposure to BaP may exert adverse effects on the immune system of broilers which may increase their susceptibility to disease, and Ns supplementation significantly reduces these alterations
- 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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