Histology and histopathology Vol.26, nº6 (2011)

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Open Access
Calcium, 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, James
A 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.
Open Access
Cytoglobin expression of rectal subepithelial myofibroblasts: Significant alterations of cytoglobin+ stromal cells in long-standing ulcerative colitis
(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Okayasu, Isao; Mikami, Tetuo; Yoshida, Tsutomu; Hana, Kiyomi; Yokozawa, Yokozawa; Sada, Miwa; Fujiwara, Mutsunori; Kawada, Norifumi
Cytoglobin/stellate cell activation-associated protein (Cygb/STAP), a hemoprotein, functions as part of an O2 reservoir with protective effects against oxidative stress in hepatic stellate cells. Heterogeneous expression of the neural cell adhesion molecule (NCAM)+ and/or α-smooth muscle actin (αSMA)+ has been noted in subepithelial myofibroblasts and interstitial cells of the same lineage in the colorectum. We have demonstrated that early genomic instability of both epithelial and stromal cells in ulcerative colitis (UC) is important for colorectal tumorigenesis, as well as for mucosal remodeling. To further clarify possible roles of stromal cells in mucosal remodeling and tumor development in UC, we here focused on Cygb expression of subepithelial myofibroblasts and interstitial cells, as well as αSMA and HSP47. Noncancerous mucosa of resected rectae from UC patients with or without colorectal neoplasia (14 and 20 cases, respectively) and of sporadic rectal cancer cases (16) was analyzed immunohistochemically, as well as by immuno-fluorescence and electron microscopy. The results, heterogeneous phenotypes of Cygb+, αSMA+ and HSP47+ subepithelial myofibroblasts and interstitial cells, corresponding to rectal stellate cells, were demonstrated. A decrease of Cygb+ subepithelial myofibroblasts and an increase of αSMA+ interstitial cells were significant in UC, as compared to normal rectal mucosa. Furthermore, a decrease of Cygb+ subepithelial myofibroblasts, correlating with αSMA+ and HSP47+ cells, was significant in long-standing UC with neoplasia. In conclusion, there are heterogeneous phenotypes of Cygb+, αSMA+ and HSP47+ subepithelial myofibroblasts and interstitial cells in the rectal mucosa. Mucosal remodeling with alterations of Cygb+ and/or αSMA+/HSP47+ stromal cells might have some relation to UC-associated tumorigenesis.
Open Access
Excavation 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.
Open Access
Cell-type specific regulation of galectin-3 expression by glucocorticoids in lung Clara cells and macrophages
(2011) Maldonado, Cristina A.; Sundblad, Victoria; Salatino, Mariana; Elia, Jorge; García, Luciana N.; Leimgruber, Caraolina; Croci, Diego O.; Rabinovich, Gabriel A.
Bronchiolar Clara cells are integral components of lung homeostasis, predominantly distributed in distal airways. In addition to the 16 kDa Clara cell protein, a major secretory product with anti-inflammatory effects, rat Clara cells express the glycan-binding protein galectin-3 and secrete it into the airways. Given the essential role of galectin-3 in the control of inflammation and the well-established function of glucocorticoids (GCs) in lung physiology, here we investigated whether galectin-3 is a target of the regulatory effects of GCs. Adult male rats were subjected to bilateral adrenalectomy and the lungs were processed for light and transmission electron microscopy, immunoelectron microscopy and Western blot analysis. Profound changes in bronchiolar Clara cells and macrophage morphology could be observed by electron microscopy after adrenalectomy. While specific galectin-3 staining was detected in the nucleus and cytoplasm of Clara cells and macrophages from control animals, cytoplasmic galectin-3 expression was dramatically reduced after adrenalectomy in both cell types. This effect was cell-specific as it did not affect expression of this lectin in ciliated cells. After dexamethasone treatment, galectin-3 expression increased significantly in the nucleus and cytoplasm of macrophages and Clara cells. Western blot analysis showed a clear decrease in galectin-3 expression in ADX animals, which was recovered after a 7-day treatment with dexamethasone. In peritoneal macrophages, galectin-3 expression was also dependent on the effects of GCs both in vivo and in vitro. Our results identify a cell type-specific control of galectin-3 synthesis by GCs in lung bronchiolar Clara cells and interstitial macrophages, which may provide an alternative mechanism by which GCs contribute to modulate the inflammatory response.
Open Access
Visualisation 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.
Open Access
Overexpression of GRP78 and GRP94 is involved in colorectal carcinogenesis
(Editores F. Hernandez y Juan F. Madrid. Murcia, Universidad de Murcia, Departamento de Biologia Celular e Histologia, 2011) Takahashi, Hiroyuki; Wang, Jian-ping; Zheng, Hua-Chuan; Masuda, Shinji; Takano, Yasuo
Glucose-related proteins (GRPs) are ubiquitously expressed in the endoplasmic reticulum and assist in protein folding and assembly, consequently considered to be molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in samples taken from several different malignant tissues. To clarify the roles of both molecules in tumorigenesis and progression of colorectal carcinomas, immunohistochemistry (IHC) was performed on tissue microarrays containing colorectal carcinomas, adenomas and the non-neoplastic mucosa (NNM) using antibodies against GRP78 and GRP94. Their expression was correlated with the clinicopathological parameters of carcinomas. Both proteins were also studied in colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) by IHC and Western blot. There was a gradually increased GRP78 expression from colorectal NNMs, carcinomas, to low-grade and high-grade adenomas (P<0.05), while up-regulated GRP94 expression from NNM, low-grade adenoma, high-grade adenoma, to carcinoma (P<0.05). The expression was similar in all the carcinoma cell lines. GRP78 expression was negatively correlated with lymphatic invasion or low GRP94 expression of the carcinomas (P<0.05), while there was no correlation of GRP94 expression with other parameters of carcinomas (P>0.05). Multivariate analysis showed that venous invasion, lymph node metastasis and UICC staging (P<0.05), but not age, sex, tumor size, differentiation, depth of invasion, lymphatic invasion, GRP78 and GRP94 expression (P>0.05), were independent prognostic factors for carcinomas. It is suggested that up-regulated expression of GRP78 and GRP94 could possibly be involved in the pathogenesis of colorectal carcinomas.
Open Access
Expression 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-gang
Cyclin 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.
Open Access
Uptake of silver from metallic silver surfaces induces cell death and a pro-inflammatory response in cultured J774 macrophages
(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Locht, Linda J.; Smidt, Camille; Rungby, Jørgen; Stoltenberg, Meredin; Larsen, Agnete
In clinical medicine metallic silver is used as anti-bacterial coating on various catheters, bandages and prostheses. By means of dissolucytosis, i.e. extracellular macrophage-mediated bio-liberation of metal ions, silver ions are continuously liberated from silver surfaces starting within minutes of exposure. The present study investigates how bio-liberation and subsequent cellular uptake of silver ions affects cell viability and cell signalling within the first 3-24 hours of exposure when J774 macrophages are grown directly on a silver surface. Autometallography (AMG) was applied to demonstrate cytoplasmatic silver uptake and localisation after 1, 3, 12 and 24 hours of exposure to metallic silver. From 12 hours onwards the cells were completely filled with silver enhanced silver-sulphur nanocrystals (AMG-silver grains). At the ultrastructural level, the silver accumulations were located to lysosome-like structures. An immunoassay cell death kit found silver-induced apoptosis after 12 and 24 hours of exposure. Necrosis was seen at the same times. Judged by mRNA analysis silver exposure statistically significantly induces TNF-α and m-CSF gene expression, especially at 3 hours. Furthermore, anti-inflammatory IL-10 transcription is reduced by silver uptake and 24 hours of silver exposure induces massive iNOS-2 gene expression. At the same time silver exposure increases the gene expression of metallothionein (MT-I/MT-II), a cystein-rich protein known for its role in detoxifying heavy metals. Our data suggest that silver ions liberated from metallic silver surfaces accumulate in lysosomes, reduce macrophage viability by apoptosis and necrosis and induce a proinflammatory response.
Open Access
Expression of smoothelin and smooth muscle actin in the skin
(Editores F. Hernandez y Juan F. Madrid. Murcia, Universidad de Murcia, Departamento de Biologia Celular e Histologia, 2011) Aneiros Fernández, José; Husein-ElAhmed, Husein; Arias-Santiago, Salvador; Campos, Antonio; Carriel, Víctor; Sánchez-Montesinos, Indalecio; Garcia del Moral, Raimundo; Sánchez, Guillermo; O’Valle, Francisco; Aneiros, J.
Introduction: Smoothelin is a cytoskeletal protein of differentiated smooth muscle cells with contractile capacity, distinguishing it from other smooth muscle proteins, such as smooth muscle actin (SMA). Objective: To evaluate the expression of smoothelin and SMA in the skin in order to establish specific localizations of smoothelin in smooth muscle cells with high contractile capacity and in the epithelial component of cutaneous adnexal structures. Methods: Immunohistochemical analysis (smoothelin and SMA) was performed in 18 patients with normal skin. Results: SMA was expressed by the vascular structures of superficial, deep, intermediate and adventitial plexuses, whereas smoothelin was specifically expressed in the cytoplasm of smooth muscle cells of the deepest vascular plexus and in no other plexus of the dermis. The hair erector muscle showed intense expression of smoothelin and SMA. Cells with nuclear expression of smoothelin and cytoplasmic expression of SMA were observed in the outer root sheath of the inferior portion of the hair follicles and intense cytoplasmic expression in cells of the dermal sheath to SMA. Conclusions: We report the first study of smoothelin expression in normal skin, which differentiates the superficial vascular plexus from the deep. The deep plexus comprises vessels with high contractile capacity, which is important for understanding dermal hemodynamics in normal skin and pathological processes. We suggest that the function of smoothelin in the outer root sheath may be to enhance the function of SMA, which has been related to mechanical stress.
Open Access
Role 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, Matthias
Background: 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.
Open Access
Efficacy 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
Open Access
Induction 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, Kunihisa
To 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.
Open Access
Laminin isoforms in atherosclerotic arteries from mice and man
(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Rauch, Uwe; Saxena, Amit; Lorkowski, Stefan; Rauterberg, Jürgen; Björkbacka, Harry; Durbeej, Madeleine; Hultgårdh-Nilsson, Anna
The properties of the arterial vasculature depend to a large extent on the activities of smooth muscle cells, which, in turn, are determined by their extracellular environment. During pathological conditions, such as atherosclerosis, this interaction is altered. In close proximity to medial smooth muscle cells are basement membrane components, such as different isoforms of laminin. These proteins can have great impact on cellular function via interaction with cell surface integrins. However, knowledge of laminins in smooth muscle cell basement membranes during normal and pathological conditions is scarce. Therefore, we have analyzed the presence of laminin isoforms in atherosclerotic lesions of apolipoprotein E (ApoE)-deficient mice. Our study revealed that the laminin chain isotype composition within atherosclerotic plaque tissue was different from the chain composition in the media. In addition, obvious differences in laminin chain composition could be observed in areas of the media, which were or were not associated with plaque tissue. Our major findings demonstrate that laminin gamma3 was exclusively present in media associated with plaque tissue. Laminin alpha2 was also enriched in these medial areas. Plaque tissue was predominantly enriched in laminin alpha5 chains. This general distribution applied to lesions both with and without a fibrous cap-like structure. The differential distribution of laminin chains were partially accompanied by changes in the presence of the integrin alpha subunits 7 and V. The distribution of laminin chains in human atherosclerotic arteries, with different size and morphology, grossly resembled their distribution in mouse arteries.

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