Histology and histopathology Vol.30, nº3 (2015)
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- PublicationOpen AccessCellular senescence in biliary pathology: Special emphasis on expression of a polycomb group protein EZH2 and a senescent marker p16INK4a in bile ductular tumors and lesions(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Sasaki, Motoko; Nakanuma, YasuniA subgroup of intrahepatic cholangiocarcinoma and combined hepatocellular- cholangiocarcinoma contain a component of cholangiolocellular carcinoma, which is composed of small bile ductular cells. Ductular reaction, a reactive lesion at the portal tract interface comprising increased bile ductules, is frequently seen in chronic advanced liver diseases. Bile duct adenoma, a benign tumor/tumorous lesion is also composed of bile ductular cells. Differential diagnosis among these bile ductular tumors/lesions is sometimes difficult. Given overexpression of a polycomb group protein EZH2 in intrahepatic cholangiocarcinoma and high expression of senescence-associated p16INK4a in ductular reactions, we plan to apply immunostaining for EZH2 and p16INK4a for differential diagnosis of these bile ductular tumors/lesions. The expression of EZH2 was seen in all cases of cholangiolocellular carcinomas, while it was not observed in bile duct adenomas or ductular reactions. In contrast, the expression of p16INK4a was seen in most bile duct adenomas and all ductular reactions, whereas it was barely seen in cholangiolocellular carcinomas. A borderline between cholangiolocellular carcinoma and the surrounding ductular reaction was clearly highlighted by the reverse expression pattern of EZH2 and p16INK4a. In conclusion, immunostaining for EZH2 and p16INK4a may be useful for differential diagnosis for bile ductular tumors/lesions.
- PublicationOpen AccessThe cellular homeostasis of the gut: what the Drosophila model points out(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Pasco, Matthieu Y.; Loudhaief, Rihab; Gallet, ArmelThe digestive tract is subjected to many aggressions throughout animal life. Since disruptions of gut physiology impact on animal fitness and survival, maintenance of gut integrity and functionality is essential for the individual. Over the last 40 years, research on rodents has aimed at understanding how cellular homeostasis of the digestive tract is maintained when challenged with disruptions. Following the discovery of stem cells in the digestive tract of Drosophila, a flurry of studies made an important contribution to our understanding of how the proliferation and the differentiation of these cells are controlled and participate in the renewal of the digestive tract. Insights into these mechanisms in Drosophila have revealed many similarities with mammalian intestinal stem cells. For instance, the highly conserved EGFR, JAK/STAT, Wingless/Wnt, Hedgehog, Integrins, BMP/TGFβ, Hippo and Insulin pathways all participate in adult intestinal cellular homeostasis. Here, we provide a literature review of recent advances in the field highlighting the adult Drosophila midgut as a convenient model for dissecting mechanisms involved in the maintenance of the cellular homeostasis of the digestive tract in conventionally reared conditions. In addition, we shed light on recently published data putting Drosophila forward as a genetic tool to decipher the mechanisms underlying intestinal diseases and intestinal tumour progression.
- PublicationOpen AccessLoss of Nm23 is associated with a more favorable tumor microenvironment in patients with breast cancer(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Durán, Esther; Cárdenas, José Miguel; Reina, Miguel Ángel; Arriazu, RiánsaresAIM: Nm23 is a metastasis suppressor gene whose downregulation triggers metastatic progression. The aim of this study was to investigate the expression of Nm23 in breast carcinomas and its relationship with tumor microenvironment markers. Methods: A retrospective study was done (128 breast cancer patients from 2007 to 2010). Nm23, LPA1, SMA, CD34, CD8, and CD68 protein expressions were evaluated using immunohistochemistry. Image analysis was used to determine the immunostaining percentage area of Nm23, LPA1, and SMA; the number of the total vessel fraction CD34 positive; and the number of CD8+ and CD68+ cells. The mean ± SE was calculated. The differences among groups were evaluated using Student t-test for parametric data and Mann Whitney U test for nonparametric data. Results: Cases were divided into two groups: Nm23+ and Nm23-. LPA1 immunostaining was significantly increased in Nm23- group. Immunostaining percentage area of SMA was not significantly higher when Nm23 was negative. CD34 immunopositive blood vessels, number of T CD8+ cells, and the number of macrophage CD68+ cells were increased when Nm23 was absent. Conclusion: Our results suggest that the absence of Nm23 causes an increase in LPA1, CD8+ and CD68+ inflammatory cells, and angiogenesis marker. Therefore, Nm23 loss could be associated with a more favorable environment for the development and dissemination of breast cancer. However, more studies are needed to determine this association.
- PublicationOpen AccessPluripotent stem cells isolated from umbilical cord form embryonic like bodies in a mesenchymal layer culture(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Tsagias, Nikos; Kouzi-Koliakos, Kokkona; Karagianis, Vasileios; Tsikouras, P.; Koliakos, George G.Recently the matrix of umbilical cord began to use as an alternative source of stem cells additionally to the blood of umbilical cord. Umbilical cord has been used mainly for mesenchymal stem cell banking. The immunological characteristics of mesenchymal stem cells in combination with their ability to avoid rejection make them an attractive biological material for transplantations. In this study the isolation of small in size pluripotent stem cells from umbilical cord expressing early transcription factors with characteristics that resemble to embryonic stem cells is investigated. Pluripotent stem cells were isolated from human umbilical cords, by a new strategy method based on unique characteristics such as the small size and the positivity on early transcription factors OCT and Nanog. An enriched population of CXCR4+ OCT+ Nanog+ CD45- small stem cells from the cord was isolated. This fraction was able to create alkaline phosphatase positive like spheres forms in a mesenchymal layer with multilineage differentiation capacity. Our results were assessed by RT PCR and electophoresis for the pluripotent genes. These data suggest that umbilical cord provides an attractive source not only of mesenchymal stem cells but moreover of pluripotent stem cells. The method described herein should be applied in the field of stem cell banking in addition to the classical umbilical cord harvesting method. Isolation of a population of cells with pluripotent characteristics from umbilical cord. Adoption of a second centrifugation step for the pluripotent stem isolation. Increasing the value of the cord and explaining the pluripotency. This work will enhance the value of umbilical cord harvesting.
- PublicationOpen AccessCurrent understanding of orofacial tissue derived mesenchymal stem cells: an immunological perspective(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Ding, Gang; Niu, Jianyi; Wei, FulanMesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells able to differentiate into multiple lineages, holding the potential for replacing damaged and diseased tissues by tissue regeneration and immunomodulatory functions. So far, MSCs have been successfully isolated and characterized from a variety of orofacial tissues, including dental pulp, periodontal ligament, root apical papilla, gingiva, etc. In addition to their self-renewal and multipotent differentiation properties, these orofacial tissue derived MSCs are also capable of profound immunomodulatory effects in vitro and in vivo, thus providing a foundation for their utilization in allogeneic application and in treating autoimmune diseases and inflammatory disorders. In this paper, we will review the current research progress of the immunomodulatory properties of orofacial tissue derived MSCs and the underlying mechanisms, emphasizing the effect of these MSCs on immune cells, which will facilitate the use of such cells in clinical treatment.
- PublicationOpen AccessDoes coenzyme-Q have a protective effect against atorvastatin induced myopathy? A histopathological and immunohistochemical study in albino rats(2015) Khalil, Mahmoud Salah; Khamis, Nehal; Al-drees, Abdulmajeed; Abdulghani, Hamza MohammadIntroduction. In addition to their lipidlowering effect, statins have pleiotropic effects that may extend their use to the treatment and prevention of various other diseases such as cancer, osteoporosis, multiple sclerosis, rheumatoid arthritis, type 2 diabetes, and Alzheimer’s disease. Consequently, the number of patients taking statins is expected to increase. A side effect of statins, statin-induced myopathy, which may result from reduced muscular coenzyme Q10 levels, limits their use. The current study investigates if supplementing with CoQ10 could ameliorate statin induced myopathy. Materials and Methods. Forty adult male albino rats were randomized into 4 groups, with 10 rats per group. The following was administered to the rats using oral gavage for 4 weeks: Group 1: 2 ml of 0.5% carboxymethyl cellulose once daily. Group 2: 100 mg/kg/ day coenzyme Q10 dissolved in 2 ml of cotton seed oil. Group 3: 10 mg/kg once daily atorvastatin dissolved in 0.5% carboxymethyl cellulose. Group 4: concomitantly received CoQ10 and atorvastatin similar to groups 2 and 3 respectively. Plasma creatine kinase levels were measured by using spectrophotometer. The right extensor digitorum longus muscle sections were stained for histological (Haematoxylin & Eosin, Masson trichrome and Phosphotungstic acid haematoxylin) and immunohistochemical (cytochrome C and Bax) examinations. Quantitative measures of cytochrome C and Bax were carried out using image analyzer. Results. Atorvastatin induced increased total creatine kinase, skeletal muscle variations in the sizes and shapes, necrosis, disorganization, nuclear pyknosis, karyorrhexis, karyolysis, dismantled plasma membrane, excess collagen fibers and lipid deposition in addition to loss of cross striation. Atorvastatin increased the intensity of the immune-positive reactions of cytochrome C and Bax. These changes were ameliorated by concomitantly giving coenzyme Q10. Conclusion: CoQ10 may ameliorate atorvastatin induced skeletal muscle injury.
- PublicationOpen AccessN-cadherin, beta-catenin and connexin 43 expression in astrocytic tumours of various grades(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Reszec, J.; Szkudlarek, M.; Hermanowicz, A.; Bernaczyk, P.S.; Mariak, Z.; Chyczewsk, L.Introduction: Astrocytic tumors are the most common primary brain tumors, but little is known about their etiology and prognostic factors. N-cadherin and beta-catenin are adhesive proteins, and are often overexpressed in many types of cancers, including breast or colorectal cancer, resulting in better prognosis. Connexin 43 is a gap junction protein involved in cellcell signaling pathway taking part in the process of carcinogenesis. The aim of the study was to evaluate Ncadherin, beta-catenin and connexin 43 expression in astrocytic tumors of various grades. Materials and methods: We examined 131 cases of astrocytic tumors, including 26 cases of diffuse astrocytoma (group I), 44 anaplasic astrocytomas (group II) and 61 glioblastoma cases (group III) - primary and secondary. To evaluate N-cadherin, beta-catenin and connexin 43 expression, we used immunohistochemical reaction with specific antibodies (Santa Cruz Biotechnology). The obtained results were correlated with clinical and morphological features. Results: Beta-catenin expression was observed in 69.3% of diffuse astrocytomas, 75% of anaplastic astrocytomas, and 82% of glioblastoma cases. Ncadherin expression was observed in 92.3% of diffuse astrocytomas, 90.1% of anaplastic astrocytomas, and in all glioblastoma cases. Connexin 43 was observed in 76.9% of diffuse astrocytomas, and in all cases of anaplastic astrocytomas and glioblastomas. Beta-catenin expression was significant within the nucleus of neoplastic cells in groups I and II. In group III, staining was observed only in the cellular membranes. Ncadherin and connexin 43 expression was observed only in the cells’ membranes. In glioblastomas, both primary and secondary, all protein expression was significant within the cells surrounding the necroses and blood vessels and weak or absent in the tumor’s margins. Conclusion: Our study shows that beta-catenin nuclear expression in group of diffuse astrocytomas and anaplastic astrocytomas is evidence for transcriptional function of beta-catenin in those groups. Strong Ncadherin and connexin 43 expression in those groups may be evidence for their role in tumor formation and progression. However, in glioblastomas a very important role of all examined proteins is generating intracellular connections to facilitate the escape of tumor cells from the effects of hypoxia or their accumulation around the blood vessels rather than tumor invasion into the brain parenchyma.
- PublicationOpen AccessAn effective and practical immunohistochemical protocol for bone specimens characterized by hyaluronidase and pepsin predigestion combined with alkaline phosphatase-mediated chromogenic detection(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Li, Shangfu; Liu, Bin; Tian, Ming; Zhang, Liangming; Tickner, Jennifer; Xu, Jiake; Rong, LiminThe aim of this study was to provide an effective procedure for immunohistochemistry (IHC) investigations of bone specimens. Samples from rat femoral and human vertebral bone were processed with a detailed and effective IHC protocol summarized here. First, a novel antigen retrieval (AR) method of hyaluronidase combined pepsin predigestion (H+P) was established and the optimal concentration and pH value for AR of bone specimens were determined. Second, the newly developed method was compared with existing AR methods (boiling in sodium citrate, hyaluronidase predigestion (H) and pepsin predigestion (P), with PBS only as the negative control) using two chromogenic detection systems (horseradish peroxidase (HRP) and alkaline phosphatase (AP)) to evaluate their efficacy in obtaining the best IHC results for bone samples. Considering the drawbacks of significant shrinking and detachment from slide for heat retrieval methods and the only moderate immunolabeling for H and P, H+P was the optimal AR method for IHC of bone specimens with the advantages of both good morphological preservation and strong immunoreactivity. Moreover, AP-mediated chromogenic detection was superior to HRP-labeled chromogenic detection due to significantly less nonspecific staining. In conclusion, we presented an effective and practical IHC protocol for bone specimens characterized by H+P predigestion combined with APmediated chromogenic detection. Finally, a detailed troubleshooting guide was provided for common mistakes that occur during IHC processing of the bone tissue samples.
- PublicationOpen AccessAge-associated murine cardiac lesions are attenuated by the mitochondria-targeted antioxidant SkQ1(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Manskikh, V.N.; Gancharova, O.S.; Nikiforova, A.I.; Krasilshchikova, M.S.; Shabalina, I.G.; Egorov, M.V.; Karger, E.M.; Milanovsky, G.E.; Skulachev, V.P.; Zinovkin, R.A.; Galkin, I.I.Age-related changes in mammalian hearts often result in cardiac hypertrophy and fibrosis that are preceded by inflammatory infiltration. In this paper, we show that lifelong treatment of BALB/c and C57BL/6 mice with the mitochondria-targeted antioxidant SkQ1 retards senescence-associated myocardial disease (cardiomyopathy), cardiac hypertrophy, and diffuse myocardial fibrosis. To investigate the molecular basis of the action of SkQ1, we have applied DNA microarray analysis. The global gene expression profile in heart tissues was not significantly affected by administration of SkQ1. However, we found some small but statistically significant modifications of the pathways related to cellto-cell contact, adhesion, and leukocyte infiltration. Probably, SkQ1-induced decrease in leukocyte and mesenchymal cell adhesion and/or infiltration lead to a reduction in age-related inflammation and subsequent fibrosis. The data indicate a causative role of mitochondrial reactive oxygen species in cardiovascular aging and imply that SkQ1 has poteential as a drug against age-related cardiac dysfunction.
- PublicationOpen AccessOcular lesions induced in infant rats by busulfan(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Saito, Tsubasa; Ando, Ryo; Ohira, Toko; Hoshiya, Toru; Tamura, KazutoshiAlthough busulfan, a bifunctional alkylating agent, is known to induce cataracts in infant rats, the full nature of busulfan-induced ocular lesions has not yet been shown. In order to clarify this point, 6-day-old rats were treated with a single dose of 20 mg/kg busulfan and the ocular tissue was histopathologically and immunohistochemically examined at 1, 2, 4, 7 and 12 days after treatment (DAT). As a result, in the nuclear layer (NL) of the peripheral retina, apoptotic cells significantly increased at 1 DAT and peaked at 2 DAT when cell proliferating activity was depressed. At 4 DAT, the NL showed wavy deformation with formation of rosette-like structures, and these changes progressed prominently at 12 DAT. In addition, a significant reduction in the retinal thickness due to decreased thickness of NL or inner NL was detected at 2 and 4 DAT. On the other hand, in the germinative zone of the lens equator, apoptotic lens epithelial cells significantly increased from 2 to 7 DAT, resulting in partial loss of lens epithelial cells at 7 and 12 DAT. At 12 DAT, prominent swelling and vacuolation of lens fibers were observed in the area from the equatorial zone to the posterior pole, indicating the development of cataract. The present results strongly suggest that prominent apoptosis in component cells was the initial and essential event underlying the developpment of busulfan-induced ocular lesions in infant rats.
- PublicationOpen AccessEmerging role of tissue lectins as microenvironmental effectors in tumors and wounds(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Smetana, Karel Jr.; Szabo, Pavol; Gál, Peter; André, Sabine; Gabius, Hans-Joachim; Kodet, Ondřej; Dvořánková, BarboraDetailed comparative analysis of at first sight not related process cascades is a means toward this aim: to trace common effector mechanisms and hereby eventually inspire innovative routes for therapeutic management. Following this concept, promotion of tumor progression by stroma, especially cancerassociated fibroblasts and smooth muscle actin-positive myofibroblasts, and beneficial activity of respective cells in wound healing have helped to delineate the involvement of endogenous lectins of the family of galectins. In addition to initiating conversion of fibroblasts to myofibroblasts, galectin-1 instructs the cells to produce a structurally complex extracellular matrix. This bioscaffold is useful for keratinocyte culture, also apparently operative in ameliorating wound healing. These functional aspects encourage to study in detail how lectin-(glycan) counterreceptor display is orchestrated. Such insights are assumed to have potential to contribute to rationally manipulate stem/precursor cells as resource in regenerative medicine.
- PublicationOpen AccessIschemic tissue injury and progenitor cell tropism: significant contributors to the pathogenesis of pterygium(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Kim, Kyoung Woo; Ha, Hyo Shin; Kim, Jae ChanPterygium is a common ocular surface disease characterized by triangular wing-like growth consisting of subconjunctival hypertrophic connective tissue. Pterygium is easily complicated by adhesion to the eyelid and diplopia related to motility restriction of the eyeball. Beyond the cosmetic problems, this condition has a catastrophic effect on quality of life. Post-surgical recurrence rates of pterygium excision have been reported to be very high. Therefore, identifying the distinct pathogenic pathways of the disease may lead to new therapeutic strategies with lower risk of treatment failure. Based on the relatively low vascularity and known-predominance of disease occurrence in the nasal conjunctiva of normal eyes, we proposed that hypoxic ischemic injury can elicit the development of pterygium. Here, we review hypoxiainducible factor (HIF)-1alpha-induced activation of the stromal cell-derived factor-1 (SDF-1)/chemokine receptor type 4 (CXCR4) signaling pathway as a possible mechanism. Supporting this concept of pathogenic mechanism, we also highlight bone marrowderived progenitor cell tropism as a main contributor to pterygium pathogenesis.