Histology and histopathology Vol.31, nº1 (2016)
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- PublicationOpen AccessThe presence of lysyl oxidase-like enzymes in human control and keratoconic corneas(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Dudakova, Lubica; Sasaki, Takako; Liskova, Petra; Palos, Michalis; Jirsova, KaterinaPurpose: Lysyl oxidases, a family comprising lysyl oxidase (LOX) and four LOX-like enzymes (LOXL1-4), catalyse the cross-linking of elastin and collagen fibrils. Keratoconus (KC) is characterized by progressive thinning leading to irregular astigmatism, resulting in significant visual impairment. Although the pathogenesis of KC remains unclear, one of the current hypotheses is based on alterations in the organization and structure of collagen fibrils. To extend existing general knowledge about cross-linking enzymes in the human cornea, in the present study we have focused on the detection of LOXL enzymes. Method: The localization and distribution of LOXL1-4 were assessed in cryosections of 7 control donors (three males and three females; 25-68 years; mean age 46±17.6 years) and 8 KC corneas (5 males and 3 females; 25-46 years; mean age 31.3±7.5 years) using indirect fluorescent immunohistochemistry (IHC). The specimens were examined using an Olympus BX51 microscope (Olympus Co., Tokyo, Japan) at a magnification of 200-1000x. Western blot analysis of 4 control and 4 KC corneas was performed for all tested enzymes. Results: All four LOX-like enzymes were present in all layers of control corneas as well as in the limbus and conjunctiva. Almost no differences between control and pathological specimens were found for LOXL1. A lower staining intensity of LOXL2 was found using IHC and Western blot analysis in KC specimens. Decreases of the signal and small irregularities in the staining were found in the epithelium, keratocytes and extracellular matrix, where a gradual anterior-posterior weakening of the signal was observed. LOXL3 IHC staining was lower in the corneal stromal extracellular matrix and keratocytes of KC samples. No prominent differences were detected using IHC for LOXL4, but a slight decrease was observed in KC corneas using Western blot analysis. Conclusion: We presume that the decrease of LOXL2 in KC corneas is more likely a consequence of the associated pathological processes (activation of stromal cells due to tissue weakening and consequent structural changes) than a direct cause leading to KC development. At this time, we are unable to provide a coherent explanation for the observed decrease of LOXL3 and LOXL4 in KC corneas.
- PublicationOpen AccessDimensions of compartments and membrane surfaces in the intact rabbit heart of importance in studies on intramyocardial transfer of blood-borne substances(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) van der Vusse, Ger J.; Verheyen, Fons; Reneman, Robert S.; Arts, TheoCardiac studies on the uptake, storage and intramyocardial transfer of blood-borne substances require detailed information on the geometric ultrastructural dimensions of myocardial compartments and parts thereof, and the membranes separating these compartments. Such a specific ultrastructural set of data of the heart is yet lacking. In the present study, we quantitatively assessed these dimensions in glutaraldehyde-perfusion fixed rabbit hearts by means of histological and tailored mathematical techniques. We showed the true ellipsoid nature of the myocardial capillary cross section and estimated the mean capillary diameter dcap. After correction for the ellipsoid shape, dcap was found to be 5.21±1.41 μm. Effective widths of the endothelial cell and the pericapillary interstitium (is1), dimensions of importance in diffusion, amounted to 187±7 and 160±10 nm, respectively. The fractional volume of the large vessels (arteries and veins larger than 10 µm), capillaries, endothelium, is1, cardiomyocytes, non-pericapillary interstitium is2, t-tubular compartment and interstitial cells amounted on average to 5.92%, 9.36%, 1.83%, 1.94%, 73.07%, 5.97%, 0.95% and 0.96%, respectively, of total myocardial volume, defined as the cardiac tissue volume, the large blood vessels included. Normalized to total myocardial volume, the surface area of the luminal and abluminal endothelial membranes and of the cardiomyocyte membrane opposing the endothelial cells amounted to 75.2±5.5x103, 82.2±6.0x103 and 89.1±6.5x103 m2/m3, respectively. The present study provides quantitative information about ultrastructural dimensions of the adult rabbit heart, among others, of importance for studies on cardiac uptake, and intramyocardial transfer and storage of blood-supplied substances.
- PublicationOpen AccessHeparanase and heparanase 2 display differently deregulation in neuroendocrine tumors, depending on their differentiation grade(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) García, Beatriz; García-Suárez, Olivia; Fernández-Vega, Iván; Vallina, Aitana; Astudillo, Aurora; Quirós, Luis M.Heparanase is a glucuronidase that appears upregulated in many human cancers and is involved in cellular invasion and tumor metastasis. Heparanase 2 is a homologue of heparanase that lacks enzymatic activity and displays anti-metastatic features. The aim of this work was to analyze the expression of both molecules in neuroendocrine tumors. We investigated the transcription of heparanases in lung neuroendocrine tumors well- and poorly differentiated using RT-PCR, and the expresion of the proteins by means of immunohistochemistry. The tumors were selected according to different malignancy WHO 2013 grades and were arranged in tissue arrays. The prometastatic enzyme heparanase appeared overexpressed in well- but not in poorly differentiated tumors, irrespective of their location. Moreover, the anti-metastatic heparanase 2 increased its expression in well-differentiated tumors, but strongly decreased in poorly differentiated ones, again independently of anatomic origin. Given the involvement of both molecules in tumor progression, through both their catalytic and non-enzymatic properties, there would seem to be a relationship between the regulation of their expression and the features of the neuroendocrine tumor.
- PublicationOpen AccessThe impact of mechanical stress on stem cell properties: The link between cell shape and pluripotency(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Caifeng Ho, Jolene; Ueda, Jun; Shimizu, Takeshiy. Embryonic development and differentiation are controlled largely by external stimuli. Mechanical forces, such as those exerted by the surrounding cells and tissues, gravity and substrate rigidity, have been shown to affect cell morphology and spreading, thus triggering signaling pathways that dictate their development. These mechanosignaling pathways play important roles in cellular differentiation and the determination of cell fate. In this review, we discuss the effects of external environmental stimuli on cell differentiation and how this affects pluripotency, as well as the key molecules and pathways involved in mechanosignaling, particularly in relation to embryonic stem cells. Advances in experimental techniques and devices used to study the different aspects of mechanobiology are also examined. Finally, the effects of mechanical stress on the initiation and maintenance of pathological processes such as cancer, as well as their implications for prognosis and possible therapies, are discussed.
- PublicationOpen AccessFrom Barrett metaplasia to esophageal adenocarcinoma: the molecular background(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Saraggi, Deborah; Fassan, Matteo; Bornschein, Jan; Farinati, Fabio; Realdon, Stefano; Valeri, Nicola; Rugge, MassimoThe molecular landscape of Barrett’s esophagus and Barrett-related neoplastic lesions is still far from being completely elucidated. Both in vitro and in vivo studies pinpointed the pathogenetic role of different morphogenic pathways (the para-homeobox, the Notch and the Sonic Hedgehog families in particular) implicated in the acquisition of the metaplastic phenotype of the esophageal mucosa. On the other hand, the most common genetic alterations observed during Barrett's carcinogenesis include disorders of major regulators of the cell cycle, as well as deregulation of the TGF-β/Smad and receptor tyrosine kinases signalling pathways. Recent comprehensive mutational profiling studies identified that the inactivation of the TP53 and of the SMAD4 tumour suppressor genes occurred in a stage-specific manner, confined to (high grade) dysplastic and neoplastic lesions, respectively. The next step will be the correlation of these findings into multidisciplinary diagnostic approaches integrating endoscopy, histology, molecular profiling and liquid biopsies. This will allow the introduction of innovative strategies for secondary prevention of esophageal adenocarcinoma based on biological rationales, and the implementation of potential novel therapeutic targets.
- PublicationOpen AccessInvolvement of lncRNA dysregulation in gastric cancer(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Sun, Ming; Nie, Feng-qi; Wang, Zhao-xia; De, Weiy. Benefiting from the fast development of sequencing technique and bioinformatics methods, more and more new long non-coding RNAs (lncRNAs) are discovered and identified. lncRNAs were firstly thought to be transcription noise that from genome desert without biological function; however, as the discovery of lncRNA XIST and HOTAIR uncovers the emerging roles of lncRNAs in development and tumorigenesis. In the past decades, accumulating evidence have indicated that lncRNAs involve in a wide range of biological functions, such as X-chromosome inactivation, reprogramming stem cell pluripotency, regulation of the immune response and carcinogenesis. Although lots of studies have demonstrated that dysregulation of lncRNAs involve in diverse diseases including cancers, the underlying molecular mechanisms of lncRNAs are not well documented. Interestingly, our previous studies and others’ have shown that numerous of lncRNAs expression was misregulated in gastric cancer. In this review, we will focus on the dysregulated lncRNAs and their biological function and underlying pathways or mechanisms in GC. Finally, we will discuss the potential roles of lncRNAs acting as biomarkers or therapeutic targets in GC patients.
- PublicationOpen AccessMammary myofibroblastoma: an update with emphasis on the most diagnostically challenging variants(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Magro, GaetanoMyofibroblastoma (MFB) is a rare benign mesenchymal tumor which usually occurs in the breast parenchyma of both females and males. Although this tumor is typically composed of bland-looking spindleshaped cells arranged in short fascicles interrupted by keloidal-like collagen fibers, several variations on this basic morphological theme do exist. With the advent of mammographic screening, an increased number of mammary MFBs are being detected and pathologists should be aware of the wide morphological and immunohistochemical spectrum exhibited by this unusual tumor. This review focuses on the most diagnostically challenging variants of mammary MFB, which could represent potential diagnostic pitfalls of malignancy, especially when evaluating needle core biopsies. In this regard the following variants of MFB, including the most recently recognized, will be presented: myxoid MFB, lipomatous MFB, epithelioid cell MFB, deciduoid cell MFB, epithelioid cell MFB with multinodular growth pattern, palisaded/ schwannian-like MFB and MFB with extensive myxoedematous stromal changes. Histological illustrations along with differential diagnostic problems for each single variant of MFB will be provided to offer helpful suggestions for a correct diagnostic approach in daily practice.
- PublicationOpen AccessATRX loss in adult supratentorial diffuse astrocytomas correlates with p53 over expression and IDH1 mutation and predicts better outcome in p53 accumulated patients(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Shao, Li-Wei; Pan, Yi; Qi, Xue-Ling; Ma, Xiao Long; Yi, Wei-Ning; Zhang, Jing; Zhong, Yan-Feng; Chang, QingBackground: IDH1/2 mutation, 1p/19qcodeletion and MGMT hypermethylation are well known molecular markers for gliomas. ATRX and p53 alterations are two lineage-specific genetic aberrations in diffuse astrocytic tumors. The aim of the present study is to clarify the significance of ATRX loss and its correlation with p53 overexpression, IDH1/2 mutations, 1p/19q-codeletion and MGMT hypermethylation in supertentorial astrocytoma, and to determine the prognostic value of these factors in Chinese patients. Methods and Results: A total of 135 adult supertentorial astrocytomas were evaluated. ATRX loss was detected by immunohistochemistry (IHC) and was shown to be much less frequent in pGBs (3.5%) than in grade II, III astrocytomas and IV sGBs (31%). Direct sequencing and/or IHC analysis of the IDH1R132H gene mutation and p53 accumulation demonstrated correlation with age. Strong correlations were found between ATRX loss and IDH1R132H mutation, p53 overexpression as well as MGMT hypermethylation. 1p/19q-codeletion detected by fluorescence in situ hybridization (FISH) showed mutually exclusive with ATRX loss and p53 accumulation. In addition, patients with p53 overexpression combined with ATRX alterations demonstrated substantially longer survival than patients with wild-type ATRX. Conclusions: There may be interactions among these distinct molecules in astrocytoma development. ATRX loss may predict better clinical outcome in astrocytoma patients with p53 overexpression as compared to patients with wild-type ATRX. Tumors with astrocytoma phenotype accompanied by 1p/19q-codeletion and IDH1R132H mutation are mutually exclusive with ATRX and p53 alterations. Routine IHC can be used for evaluation of ATRX loss, p53 protein accumulation and IDH1R132H mutation, which may allow a means of classification of astrocytoma outcome.
- PublicationOpen AccessSOX10 and Olig2 as negative markers for the diagnosis of ependymomas: An immunohistochemical study of 98 glial tumors(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Švajdler, Marián; Rychlý, Boris; Mezencev, Roman; Fröhlichová, Lucia; Bednárová, Antónia; Pataky, František; Daum, OndřejSOX10 belongs to the family of transcription factors essential for the development of neural crest, peripheral nervous system and melanocytes. It is presently used in histopathology as a marker of melanocytic differentiation. SOX10 is expressed in normal brain tissue in oligodendrocytes, but the information about SOX10 expression in primary tumors of the central nervous system is quite limited. In this study, we examined the expression of SOX10 and Olig2 by immunohistochemistry in a series of 98 glial tumors and explored their specificity and sensitivity for differential diagnosis of ependymal vs non-ependymal tumors. In addition, we examined the expression of EMA and CD99 in ependymal tumors. SOX10 and Olig2 staining were scored as negative if no positive cells or only a few positive cells (typically up to 1-3%) were found. In all other instances, SOX10 or Olig2 staining was scored as positive. Out of 44 examined ependymal tumors none was found to express SOX10 and 7 specimens showed only a few SOX10-positive cells that likely corresponded to entrapped nonneoplastic oligodendrocytes. In contrast, non-ependymal tumors expressed SOX10 in 26/54 (48%) specimens. Olig2 was positive in 5 out of 44 ependymomas (11%) and 50 out of 54 (93%) non-ependymal tumors (astrocytomas and oligodendrogliomas). EMA and CD99 expression was found in 33/44 (75%) and 11/44 (25%) of ependymomas, respectively. SOX10-positivity rules out the diagnosis of ependymoma among other glial tumors with high confidence.
- PublicationOpen AccessImmunohistochemical and radiological characterization of wound healing in porcine liver after radiofrequency ablation(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Stadlbauer, Vanessa; Lang-Olip, Ingrid; Leber, Bettina; Mayrhauser, Ursula; Koestenbauer, Sonja; Tawdrous, Monika; Moche, Michael; Sereinigg, Michael; Seider, Daniel; Iberer, Florian; Wiederstein-Grasser, Iris; Horst Portugaller, Rupert; Stiegler, PhilippBackground: Radiofrequency ablation (RFA) is a minimal invasive therapeutic option for patients with hepatocellular carcinoma or liver metastases. We investigated RFA-induced cellular changes in the liver of pigs. Material and Methods: Healthy pigs (n=18) were sacrificed between day 0 and 3 months after RFA. The wound healing process was evaluated by computed tomography (CT), chromotrope anilinblue (CAB) staining of large-scale and standard tissue sections. Immunohistochemistry (IHC) for heat shock protein 70, Caspase-3, Ki67, Reelin, Vinculin, Vimentin and αSMA was perfomed. Results: One day after RFA, CAB staining showed cell damage and massive hyperaemia. All IHC markers were predominantly expressed at the outer borders of the lesion, except Reelin, which was mainly detected in untreated liver regions. By staining for Hsp70, the heat stress during RFA was monitored, which was most distinct 1-2 days after RFA. CT revealed decreased lesion size after one week. Development of a Vimentin and α-SMA positive fibrotic capsule was observed. Conclusion: In the early phase signs of cell damage, apoptosis and proliferation are dominant. Reduced expression of Reelin suggests a minor role of hepatic stellate cells in the RFA zone. After one week myofibroblasts become prominent and contribute to the development of the fibrotic capsule. This elucidates the pathophysiology of RFA and could contribute to the future optimization of RFA procedures.
- PublicationOpen AccessEstrogen-deficient osteoporosis enhances the recruitment and activity of osteoclasts by breast cancer cells(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Salamanna, Francesca; Pagani, Stefania; Maglio, Melania; Borsari, Veronica; Giavaresi, Gianluca; Martelli, Alberto M.; Buontempo, Francesca; Fini, MilenaTo reduce the burden of bone metastases, the pathophysiology of the metastatic niche should be elucidated and targeted. The aim of the present study was to assess the effect of tumor cells on osteoclast (OC) recruitment and activity in the presence of altered bone remodelling. Peripheral blood mononuclear cells (PBMC) were isolated from healthy and ovariectomized (OVX) rats and co-cultured with MRMT-1 rat breast carcinoma cells or with their conditioned medium for 1 and 2 weeks. Alamar Blue viability test, synthesis of cathepsin K, transforming growth factor-beta 1 (TGFβ1), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-7, MMP-9, FITC-conjugate phalloidin staining and tartrate-resistant acid phosphatase (TRAP) staining were evaluated. The results indicate that breast carcinoma cells induced different responses in PBMC derived from rats affected by estrogen deficiency osteoporosis (OP) in comparison with healthy ones, with a significant increase in proliferation rate, OC differentiation, synthesis of TNF-α, MMP-7 and MMP-9. The data support the “proof of concept” that OP due to estrogen deficiency might offer a receptive site for cancer cells to form bone metastases.