Histology and histopathology Vol.29, nº11 (2014)
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- PublicationOpen AccessMALDI mass spectrometry imaging of formalin-fixed paraffin-embedded tissues in clinical research(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Gorzolka, Karin; Walch, AxelThe molecular investigation of archived formalin-fixed, paraffin-embedded (FFPE) tissue samples provides the chance to obtain molecular patterns as indicatives for treatment and clinical end points. MALDI mass spectrometry imaging is capable of localizing molecules like proteins and peptides in tissue sections and became a favorite platform for the targeted and non-targeted approaches, especially in clinical investigations for biomarker research. In FFPE tissues the recovery of proteomic information is constrained by fixation-induced cross-links of proteins. The promising new insights obtained from FFPE in combination with the comprehensive patients’ data caused much progress in the optimization of MS imaging protocols to investigate FFPE samples. This review presents the past and current research in MALDI MS imaging of FFPE tissues, demonstrating the improvement of analyses, their actual limitations, but also the promising future perspectives for histopathological and tissue-based research.
- PublicationOpen AccessINF-γ sensitizes head and neck squamous cell carcinoma cells to chemotherapy-induced apoptosis and necroptosis through up-regulation of Egr-1(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Xu, Bei; Shu, Yongqian; Liu, PengHead and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Acquired resistance to standard chemotherapy accounts for most of treatment failure. Here we demonstrate that Interferon-γ (INF-γ) may up-regulate Egr-1 gene expression in HNSCC cell line SCC-25. Forced expression of Egr-1 sensitizes SCC-25 cells to chemotherapy-induced apoptosis and necroptosis, a novel form of programmed cell death. Egr-1 upregulation also significantly increases the production of Thrombospondin-1 (TSP-1), a matricellular glycoprotein which has been described to induce cell death in HNSCC. Moreover, INF-γ-induced sensitization of cells to chemotherapy-mediated cell death and TSP-1 production could be markedly abolished by Egr-1 silencing. The present investigation provides the first evidence that INF-γ may sensitize HNSCC cells to chemotherapy-induced apoptosis and necroptosis through up-regulation of Egr-1. These data support the combination use of INF-γ and cytotoxic drugs for HNSCC Therapy.
- PublicationOpen AccessFlavonoid profile of Lupinus mexicanus germinated seed extract and evaluation of its neuroprotective effect(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Uribe-Gómez, José de Jesús; Zamora-Natera, Juan Francisco; Bañuelos-Pineda, Jacinto; Kachlicki, Piotr; Stobieck, Maciej; García-López, Pedro MacedonioThe aim of this study was to determine the flavonoid profile of Lupinus mexicanus germinated seed extract (PE) and to evaluate its effect as a phytoestrogen on the morphometric parameters of CA3 hippocampal neurons of ovariectomized rats (OVX). L. mexicanus seeds, germinated for 48 h, were homogenized and macerated using an 80% ethanol solution. The extract was analyzed by HPLC/MS-MS. Thirty young Wistar strain female rats (200±10 g) were randomly distributed into four groups: sham operated (S) treated with dimethyl sulfoxide (vehicle); ovariectomized and treated with 1250 µg of PE extract (OVX-PE); ovariectomized and treated with 5 µg estradiol benzoate (OVX-EB); and ovariectomized and vehicle treated (OVX). All substances were injected subcutaneously daily for 28 days. On day 29, the animals were sacrificed, perfused, and fixed to obtain the brains for histological processing. Each brain was cut and stained with hematoxylin and eosin. The thickness of the stratum oriens (SO), the nuclear diameter, and the neuronal density were measured in the hippocampus CA3 area. Nine different flavonoids and one non-identified compound were detected. The histological analysis demonstrated that the thickness of the SO was higher in the OVX-EB and S groups than in the OVX-PE and OVX groups (p<0.05); in addition, the nuclear diameters of the neurons in the OVX-EB and S groups were higher compared with the other groups (p<0.05). The OVX group had the highest cellular density among groups (p<0.05). Based on our results, the PE obtained did not have beneficial effects on CA3 hippocampal neurons.
- PublicationOpen AccessFactors influencing malignant evolution and long-term survival in solitary fibrous tumours of the pleura(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Rodríguez-González, Marta; Nuria M., Novoa; Gomez, Maria T.; García, J.L.; Ludeña, DoloresSolitary pleuro-pulmonary fibrous tumours are relatively uncommon neoplasms that are difficult to manage therapeutically and which, cytogenetically, have been poorly studied. The aim of the present work was to analyse the characteristics of a series of consecutive operated solitary pleural fibrous tumours in an attempt to discover a malignant pattern of evolution. This was a retrospective observational study of 19 cases. Samples were studied for clinical, histological, immunohistochemical and cytogenetic characteristics (aCGH, FISH). Descriptive statistics were used: the Kapplan-Meyer log-rank test and the Cox-regression model for survival analysis. Analysis of malignant evolution was achieved using 2x2 tables; significant factors were included in a binary logistic regression model. Parietal pleural implantation of the primary tumour, high mib1 expression, and low p53 expression were seen to be statistically significant factors for survival. We recommend a close follow-up for patients with a malignant primary tumour and low p53 expression and a regular long-term follow-up for benign primary tumours with a high mib1 index, high positive p53, and deletions. These findings need confirmation in more extensive series.
- PublicationOpen AccessPrognostic impact of perineural, blood and lymph vessel invasion for esophageal cancer(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Tachezy, Michael; Tiebe, Anne-Kathrin; Gebauer, Florian; Kutup, Asad; Tharun, Lars; Pantel, Klaus; Izbicki, Jacob Robert; Vashist, Yogesh KumarBackground: With a median survival of <22 months esophageal cancer is one of the most aggressive tumors, up to 20% of node negative patients develop systemic relapse. Studies investigating the prognostic impact of tumor-micro-invasion in blood (AI) and lymphatic vessels (LVI) as well as perineural invasion (PNI) have shown inconsistent results. The aim of the present study was to investigate the prognostic value of the aforementioned factors in a large homogenously treated cohort of patients with esophageal cancer. Methods: Data from 695 patients with surgically treated esophageal cancer were analyzed. AI, LVI and PNI were determined and data were statistically correlated with clinico-pathological parameters and survival of the patients. Results: Thirteen percent of all specimens showed an AI, 35% a LVI and 5% a PNI. The invasion factors were mostly significantly correlated with the established prognostic parameter, including bone marrow micrometastases. Kaplan-Meier analysis revealed a prognostic impact for LVI in both cancer subtypes, while AI and PNI were significant factors in adenocarcinoma only. In multivariate analysis, none showed statistical significance. However, sub-analysis of completely resected, node negative and non-metastasized patients showed a significant prognostic impact of LVI. Conclusion: The prognostic significance of AI, LVI and PNI seems to be limited compared to the established prognostic parameters of the UICC staging system. In completely resected, node negative and nonmetastasized patients, LVI is an independent prognostic parameter for a worse outcome. Those patients might benfit from additional treatment or more intensive follow up.
- PublicationOpen AccessCorrelation between 3D microstructural and 2D histomorphometric properties of subchondral bone with healthy and degenerative cartilage of the knee joint(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Lahm, Andreas; Kasch, Richard; Spank, Heiko; Erggelet, Christoph; Esser, Jan; Merk, Harry; Mrosek, EikeCartilage degeneration of the knee joint is considered to be a largely mechanically driven process. We conducted a microstructural and histomorphometric analysis of subchondral bone samples of intact cartilage and in samples with early and higher- grade arthritic degeneration to compare the different states and correlate the findings with the condition of hyaline cartilage. These findings will enable us to evaluate changes in biomechanical properties of subchondral bone during the evolution of arthritic degeneration, for which bone density alone is an insufficient parameter. From a continuous series of 80 patients undergoing implantation of total knee endoprosthesis 30 osteochondral samples with lesions macroscopically classified as ICRS grade 1b (group A) and 30 samples with ICRS grade 3a or 3b lesions (group B) were taken. The bone samples were assessed by 2D histomorphometry (semiautomatic image analysis system) and 3D microstructural analysis (high-resolution microCT system). The cartilage was examined using the semiquantitative real-time PCR gene expression of collagen type I and II and aggrecan. Both histomorphometry and microstructural and biomechanical analysis of subchondral bone in groups A and B consistently revealed progressive changes of both bone and cartilage compared with healthy controls. The severity of cartilage degeneration as assessed by RT PCR was significantly correlated with BV/TV (Bone Volume Fraction), Tb.Th (Trabecular Thickness) showed a slight increase. Tb.N (Trabecular Number), Tb.Sp (Trabecular separation) SMI (Structure Model Index), Conn.D (Connectivity Density) and DA (Degree of Anisotropy) were inversely correlated. We saw sclerotic transformation and phagocytic reticulum cells. Bone volume fraction decreased with an increasing distance from the cartilage with the differences compared with healthy controls becoming greater in more advanced cartilage damage. The density of subchondral bone alone is considered an unreliable parameter for classifying changes evolving over time. The progressive damage of subchondral bone seen in the present study correlates well with cartilage changes. Trabecular orientation is also impaired, which explains the changes in biomechanical parameters and the inadequate load transfer and excessive loading of cartilage. Besides subchondral bone density, which in turn correlates with cartilage thickness, other parameters such as structure model index and grade of anisotropy best reflect mechanical properties such as Young modulus, compressive strength, tensile stress, and failure energy. However, it remains unclear whether the mechanical interaction of the mineralized subchondral tissues with articular cartilage works vice versa. The possibility of a biochemical signalling from the degenerating cartilage via the synovial fluid and bone- cartilage crosstalks via subchondral pores may indeed explain a certain depth dependency of subchondral bone changes.
- PublicationOpen AccessRole of discoidin domain receptor 2 in wound healing(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Márquez, Joana; Olaso, ElviraUntil recently, collagen interactions with cells had been ascribed to integrins. The identification of the Discoidin Domain Receptor (DDR) family as collagen receptors represents a new paradigm in the regulation of collagen-cell interactions. How DDR signaling is biochemically linked to specific cell regulatory functions remains largely unknown. Moreover, the characteristic slow and substained phosphorylation of DDRs and the elevated expression of DDR2 in the myofibroblasts of healing wounds suggest a role for DDR2 in physiological and pathological wound healing. In fact, DDR2 signaling regulates cell proliferation and extracellular matrix synthesis, which are key aspects of fibroblast contribution to tissue healing. In this review we summarize evidence in favor of this concept.
- PublicationOpen AccessEffect of prior exercise training and myocardial infarction-induced heart failure on the neuronal and glial densities and the GFAP-immunoreactivity in the posterodorsal medial amygdala of rats(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Salazar, Ana Paula S.; Quagliotto, Edson; Alves, Jadson; Oliveira, Fernando A.; Saur, Lisiani; Xavier, Léder L.; Pagnussat, Aline S.; Rasia-Filho, AlbertoExercise training has neuroprotective effects whereas myocardial infarction (MI) and heart failure (HF) can cause neuronal death and reactive gliosis in the whole amygdala. The posterodorsal medial amygdala (MePD) is involved with cardiovascular reflexes and the central control of sympathetic/parasympathetic responses. Our aim was to study the effects of prior exercise training and of MI-induced HF on the neuronal and glial densities and the glial fibrillary acidic proteinimmunoreactivity (GFAP-ir) in the MePD of adult male rats. Animals (n=5/group) were: control, sedentary submitted to a sham MI (Sed Sham), sedentary submitted to MI/HF (Sed HF), trained on a treadmill and submitted to a sham MI (T Sham) or trained on a treadmill and submitted to MI/HF (T HF). The number of neurons and glial cells in the MePD was estimated using the optical fractionator and the GFAP-ir was quantified by optical densitometry. In the respective groups, treadmill training improved physical performance and MI damaged near 40% of the left ventricle. There was a hemispheric lateralization effect on the density of neurons (higher in the right MePD), but no significant difference in either the neuronal or the glial densities due to experimental condition. Regional GFAP-ir results revealed that the Sed HF group had a higher expression in the left MePD compared to the control and the Sed Sham rats (p<0.01). The present data did not evidence the effects of training or MI/HF in the MePD cellular density, but indicate a possible local restructuring of astrocytic cytoskeleton after MI/HF in rats.
- PublicationOpen AccessEpithelial mesenchymal transition in the progression of renal disease in dogs(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Benali, S.L.; Lees, G.E.; Castagnaro, M.; Aresu, L.Chronic kidney disease (CKD) in dogs is the final common pathway resulting from persistent renal injury and is characterized by progressive tubulointerstitial damage (TID). Pathogenesis of CKD is divided into an initial inflammatory phase with a predominantly mononuclear infiltrate followed by a fibrotic phase with increased numbers of fibroblasts and extracellular matrix deposition that causes a progressive reduction of functional parenchyma. Proteinuria is a common manifestation of renal diseases in dogs, and its role in the pathogenesis of CKD is still uncertain. Nevertheless, the degree of proteinuria in dogs correlates with TID progression. Increased protein filtration may have direct effects on tubular epithelial cells (TECs) that induce them to express the major histocompatibility complex type II, and thereby contribute to lymphocyte recruitment. Thus, an active pro-inflammatory role is proposed for TECs in TID progression. Moreover TECs are believed to actively participate in the mechanisms of renal fibrosis. Epithelial-Mesenchymal-Transition (EMT) of TECs in canine TID has been studied in the last decade. Downregulation of adhesion molecules and loss of epithelial markers in TECs directly correlate with the severity of TID and with de novo expression of mesenchymal markers. Tubular basement membrane (TBM) disruption is an early EMT event. Increased activity of Matrix Metalloproteinase-2 and its co-localization with TBM splitting suggests an active role for the enzyme in inducing EMT. Processes occurring in canine CKD share many similarities with its human counterpart, making the dog a good model in which to examine the mechanisms of TID progression.
- PublicationOpen AccessEvidence for different expression profiles for c-Met, EGFR, PTEN and the mTOR pathway in low and high grade endometrial carcinomas in a cohort of consecutive women. Occurrence of PIK3CA and K-Ras mutations and microsatellite instability(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Thoury, Anne; Descatoire, Véronique; Kotelevets, Larissa; Kannengiesser, Caroline; Bertrand, Guylène; Theou-Anton, Nathalie; Frey, Caroline; Genestie, Catherine; Raymond, Eric; Chastre, Eric; Lehy, Thérèse; Walker, FrancineMolecular and genetic investigations in endometrial carcinogenesis may have prognostic and therapeutic implications. We studied the expression of EGFR, c-Met, PTEN and the mTOR signalling pathway (phospho-AKT/phospho-mTOR/phospho-RPS6) in 69 consecutive tumours and 16 tissue microarrays. We also analysed PIK3CA, K-Ras mutations and microsatellite instability (MSI). We distinguished two groups: group 1 (grade 1 and 2 endometrioid cancers) and group 2 (grade 3 endometrioid and type II clear and serous cell cancers). We hypothesised that these histological groups might have different features. We found that a) survival was higher in group 1 with less aggressive tumours (P<0.03); b) EGFR (P=0.01), PTEN and the AKT/mTOR/RPS6 signalling pathway were increased in group 1 versus group 2 (P=0.05 for phospho-mTOR); c) conversely, cMet was higher (P<0.03) in group 2 than in group 1; d) In group 1, EGFR was correlated with c-Met, phosphomTOR, phospho-RPS6 and the global activity of the phospho-AKT/phospho-mTOR/phospho-RPS6 pathway. In group 2, EGFR was correlated only with the phosphoAKT/phospho-mTOR/phospho-RPS6 pathway, whereas c-Met was correlated with PTEN; e) survival was higher for tumours with more than 50% PTEN-positive cells; f) K-RAS and PIK3CA mutations occurred in 10-12% of the available tumours and MSI in 40.4%, with a loss of MLH1 and PMS2 expression. Our results for endometrial cancers provide the first evidence for a difference in status between groups 1 and 2. The patients may benefit from different targeted treatments, antiEGFR agents and rapamycin derivatives (anti-mTOR) for group 1 and an anti c-MET/ligand complex for group 2.
- PublicationOpen AccessBiomaterial scaffolds used for the regeneration of spinal cord injury (SCI)(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Kim, Moonhang; Park, So Ra; Choi, Byung HyuneThis review presents a summary of various types of scaffold biomaterials used alone or together with therapeutic drugs and cells to regenerate spinal cord injury (SCI). The inhibitory environment and loss of axonal connections after SCI give rise to critical obstacles to regeneration of lost tissues and neuronal functions. Biomaterial scaffolds can provide a bridge to connect lost tissues, an adhesion site for implanted or host cells, and sustained release of therapeutic drugs in the injured spinal cord. In addition, they not only provide a structural platform, but can play active roles by inhibiting apoptosis of cells, inflammation and scar formation, and inducing neurogenesis, axonal growth and angiogenesis. Many synthetic and natural biomaterial scaffolds have been extensively investigated and tested in vitro and in animal SCI models for these purposes. We summarized the literature on the biomaterials commonly used for spinal cord regeneration in terms of historical backgrounds and current approaches.
- PublicationOpen AccessRole of skeletal muscle in mandible development(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Rot, Irena; Mardesic-Brakus, Snjezana; Costain, Willard J.; Saraga-Babic, Mirna; Kablar, BorisAs a continuation of the previous study on palate development (Rot and Kablar, 2013), here we explore the relationship between the secondary cartilage mandibular condyles (parts of the temporomandibular joint) and the contributions (mechanical and secretory) from the adjacent skeletal musculature. Previous analysis of Myf5-/- :MyoD-/- mouse fetuses lacking skeletal muscle demonstrated the importance of muscle contraction and static loading in mouse skeletogenesis. Among abnormal skeletal features, micrognathia (mandibular hypoplasia) was detected: small, bent and posteriorly displaced mandible. As an example of Waddingtonian epigenetics, we suggest that muscle, in addition to acting via mechanochemical signal transduction pathways, networks and promoters, also exerts secretory stimuli on skeleton. Our goal is to identify candidate molecules at that muscle-mandible interface. By employing Systematic Subtractive Microarray Analysis approach, we compared gene expression between mandibles of amyogenic and wild type mouse fetuses and we identified up- and downregulated genes. This step was followed by a bioinformatics approach and consultation of webaccessible mouse databases. We searched for individual tissue-specific gene expression and distribution, and for the functional effects of mutations in a particular gene. The database search tools allowed us to generate a set of candidate genes with involvement in mandibular development: Cacna1s, Ckm, Des, Mir300, Myog and Tnnc1. We also performed mouse-to-human translational experiments and found analogies. In the light of our findings we discuss various players in mandibular morphogenesis and make an argument for the need to consider mandibular development as a consequence of reciprocal epigenetic interactions of both skeletal and non-skeletal compartments.