Histology and histopathology Vol.28, nº 4 (2013)
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- PublicationOpen AccessMorphometric analyses of normal pediatric brachial biceps and quadriceps muscle tissue(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Sallum, Adriana M.E.; Varsani, Hemlata; Holton, Janice L.; Marie, Suely K.N.; Wedderburn, Lucy R.Pediatric normal brachial biceps (14 specimens) and quadriceps muscles (14 specimens) were studied by immunohistochemistry to quantify fiber-type, diameter and distribution, capillary density, presence of inflammatory cells (CD3, CD20, CD68) and expression of neonatal myosin and MHC class 1 proteins. Brachial biceps showed more fast-twitch fibers and lower capillary/fiber ratio than quadriceps. The mean diameter of both fiber types was smaller in biceps than quadriceps. Fast-fibers were smaller than slow-fibers, and capillary/fiber ratio was <1.0 in both muscles. Fiber size and capillary / fiber ratio increased with age. Normal limits for infiltrating haematopoietic cells were <4 T lymphocytes, or CD68+ cells, very few B cells, <6 neonatal myosin positive fibers, and no fibers MHC class 1 positive in one x20 field, for both muscles. The present comparison of quantitative findings between brachial biceps and quadriceps may allow standardization of the assessment of pathological changes in both pediatric muscles.
- PublicationOpen AccessHepatic response to chronic hypoxia in experimental rat model through HIF-1 alpha, activator protein-1 and NF-kappa B(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Lau, Thomas Y.H.; Xiao, Jia; Liong, Emily C.; Liao, Linchuan; Leung, Tung-Ming; Nanji, Amin A.; Fung, Man Lung; Tipoe, George L.Chronic liver diseases are commonly associated with tissue hypoxia that may cause inflammation, oxidative stress, liver cell injury and increased nuclear transcriptional regulation. The hepatic response to chronic hypoxia at the molecular level has not yet been clearly understood until now. The aim of this study is to investigate whether nuclear transcription factors [hypoxia-inducible factor-1 (HIF-1α), activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB)] exhibit activity changes during hepatic response to chronic hypoxia. Blood and liver samples were collected from adult Sprague-Dawley rats living in atmospheric air or 10% oxygen for four weeks. Levels of serum alanine aminotransferase (ALT), 8-isoprostane and nitrotyrosine were measured. The activities of nuclear transcription factors and the expression of downstream genes (iNOS, eNOS, ET-1 and VEGF) were measured using RT-PCR, Western blotting and Gel shift analysis. Results showed that serum ALT level, 8-isoprostane level and formation of nitrotyrosine were within normal range at all time-points. In the hypoxic liver, DNAbinding activities of HIF-1α, NF-κB and AP-1 increased significantly. Expression levels of iNOS, VEGF and ET1 progressively increased from day 7 to day 28. eNOS was also elevated in the hypoxic liver. In conclusion, our study suggests that increased activity of HIF-1α, AP-1 and NF-κB may partly play a significant role in the hepatic response to oxidative stress and liver injury under chronic hypoxia. The increased expression of VEGF, ET-1, iNOS and eNOS may be partly due to the compensatory mechanism in the vascular beds of the liver in response to chronic hypoxia.
- PublicationOpen AccessExpression of receptor for hyaluronan-mediated motility (RHAMM) in ossifying fibromas(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Hatano, Hiroko; Ogawa, Ikuko; Shigeishi, Hideo; Kudo, Yasusei; Ohta, Kouji; Higashikawa, Koichiro; Takechi, Masaaki; Takata, Takashi; Kamata, NobuyukiFibro-osseous lesions of the jaw are poorly understood because of a significant overlap of clinical, radiological and histological features among the various types, though they present distinct patterns of disease progression. An ossifying fibroma is associated with significant cosmetic and functional disturbances, as it shows expansive proliferation. Thus, it is important to establish a specific marker, as well as clearly elucidate its etiology for diagnosis and proper treatment. We previously established immortalized cell lines from human ossifying fibromas of the jaw and found that they highly expressed the receptor for hyaluronan (HA)-mediated motility (RHAMM). In this study, we examined the expression of RHAMM mRNA in 65 fibro-osseous lesions, including ossifying fibroma, fibrous dysplasia and osseous dysplasia, as well as 5 normal jaws, using real-time RT-PCR and immunohistochemistry assays. RHAMM mRNA and protein expression were significantly elevated in the ossifying fibroma specimens. These results suggest that detection of upregulated RHAMM expression in an ossifying fibroma assists with differential diagnosis and has a key role in elucidation of its pathophysiology.
- PublicationOpen AccessMIA - a new target protein for malignant melanoma therapy(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Schmidt, Jennifer; Riechers, Alexander; Bosserhoff, Anja-KatrinMalignant melanoma, a malignancy of pigment producing cells, causes the greatest number of skin cancer-related deaths worldwide. The tumor is characterized by its aggressive phenotype and can metastasize at very early stages of the disease. Since metastatic lesions are usually characterized by an intrinsic resistance to standard radiation and chemotherapy, the prognosis of this tumor remains very poor in advanced stages. Melanoma inhibitory activity (MIA), an 11 kDa protein expressed and secreted by melanoma cells after their malignant transformation, is known to play a key role in melanoma development, progression and tumor cell invasion. After its secretion, which is restricted to the rear pole of migrating cells, MIA protein directly interacts with cell adhesion receptors and extracellular matrix molecules. By this mechanism, MIA protein actively facilitates focal cell detachment from surrounding structures at the cell rear and strongly promotes tumor cell invasion and formation of metastases. It has further been demonstrated that MIA contributes to immunosuppression frequently seen in malignant melanomas by binding to integrin α4ß1 expressed by leukocytes and thus inhibiting cellular antitumor immune response. Analyses at the molecular level revealed that MIA protein reaches functional activity by self assembly. Functional inactivation of MIA protein by dodecapeptides that directly bind to the dimerization interface leads to a strongly reduced tumor cell invasion in an in vivo mouse melanoma model. The molecular understanding of the contribution of MIA protein to formation of metastases provides an excellent starting point for the development of a new strategy in malignant melanoma therapy.
- PublicationOpen AccessMCM2 expression levels predict diagnosis and prognosis in gastric cardiac cancer(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Liu, Min; Li, Jin-Song; Tian, Dong-Ping; Huang, Bo; Rosqvist, Seema; Su, MinBackground: Gastric Cardiac Cancer (GCC) has high incidence and poor prognosis requiring early screening of high-risk populations. Minichromosome maintenance (MCM) proteins are used as diagnosticbiomarkers in many cancers but not validated for GCC. We evaluate MCM protein 2 (MCM2), comparing it with the validated markers Ki67 and PCNA. Methods: GCC and corresponding cardiac precancerous samples were immunostained with Ki67, MCM2 and PCNA antibodies. Results: 90% of dysplasia samples expressed MCM2, whereas Ki67 and PCNA were expressed in 67% and 80% respectively. The sensitivity and negative predictive values of MCM2 were also superior at 90% and 87%, respectively. Ki67 and PCNA expression was correlated with MCM2, but their expressions seldom reached surface layers, whereas MCM2 manifested mostly in easily accessible superficial layers. Labeling indices (LI) of Ki67 and PCNA were also lower. Significant associations between LI (MCM2), LI (PCNA), and TNM-stages, lymph node metastases and GCC grade were found (P<0.05). Increased protein expressions were associated with reduced overall and disease-free survival (P<0.05). Although Ki67 and PCNA were significant prognostic factors, there was no significant improvement in multivariate statistical analyses, in contrast to LI (MCM2) findings. Conclusions: MCM2 is a sensitive, specific and efficient biomarker of GCC having potential use in clinic
- PublicationOpen AccessPregnane X receptor and human malignancy(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Koutsounas, Ioannis; Patsouris, Efstratios; Theocharis, Stamatios E.Pregnane X Receptor (PXR) is a member of the nuclear receptor superfamily, expressed in liver, intestine and other tissues. PXR exerts transcriptional regulation by binding to its DNA response elements as an heterodimer with Retinoid X Receptor (RXR). This nuclear receptor is implicated in the homeostasis of numerous endobiotics, such as glucose, lipids, steroids and bile acids. Additionally, the activation of PXR induces expression of drug metabolizing enzymes (DMEs) and transporters, including multidrug resistance protein 1 (MDR1), leading to regulation of xenobiotic metabolism and drug-drug interactions. New roles for PXR have been established in inflammatory bowel disease, bone homeostasis, liver steatosis, antifibrogenesis and oxidative stress. PXR has, additionally, a multifactorial impact on cancer, either by directly affecting cell proliferation and apoptosis or by inducing chemotherapy resistance, in colon, breast, prostate, and endometrial cancer, and in osteosarcoma. PXR polymorphisms may also have clinical significance in certain types of cancer and their treatment. Further studies are needed in order to clarify the mechanisms involved in PXR-regulated carcinogenesis. PXR downregulation could be considered as a novel therapeutic approach to overcome chemoresistance, while future research should be mainly focused on modulating PXR status in order to increase chemotherapy effectiveness and finally improve cancer patient prognosis.
- PublicationOpen AccessCancer-related microRNAs and their role as tumor suppressors and oncogenes in hepatocellular carcinoma(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Gailhouste, Luc; Ochiya, TakahiroMicroRNAs (miRNAs) have emerged as key factors involved in several biological processes, including development, differentiation, cell proliferation, and tumorigenesis. In hepatocellular carcinoma (HCC), miRNAs frequently present aberrant expression profiles, which make them potentially attractive for diagnostic or prognostic applications. Currently, accumulating evidence is indicating the role of miRNAs as tumor suppressors or oncogenes in hepatic malignancies. In particular, comprehensive studies have made possible a better understanding of HCC behavior, such as tumor growth, response to therapies, metastatic potential, or recurrence, regarding the altered expression of cancerrelated miRNAs. Based on these findings, efforts are under way to define new markers for liver cancer in both invasive (hepatic biopsy or tumor resection) and noninvasive (circulating miRNAs in blood serum) ways. Due to their implication in the control of various cell processes altered in HCC, cancer-related miRNAs also offer encouraging perspectives for the development of innovative cancer therapies. In this article, we review the importance of miRNA deregulation in HCC progression and the role of these small non-coding RNAs as tumor suppressors and oncogenes. The significance of miRNAs in HCC diagnosis and miRNA-based therapeutic strategies is then discussed.
- PublicationOpen AccessCellular and molecular mechanisms of intestinal elongation in mammals: the long and short of it(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Cervantes, SaraThe gastrointestinal tract carries out essential functions for the organism, including the digestion and absorption of nutrients. The cells lining the lumen of the gut tube derive from the endoderm, one of the three germ layers formed during gastrulation. The length of the intestinal tract determines its digestive and absorptive capacity, and so the intestine expands several times the length of the whole body to ensure an adequate absorptive area to meet nutritional demands. However, the endoderm starts out as a small sheet of cells spanning less than the whole length of the head-fold embryo. In order to achieve its final shape and size, the cells in the endoderm undergo extensive growth and profound morphogenetic changes, which are governed by embryonic signaling pathways and transcription factors. This review, based on mouse development, summarizes our current knowledge of the cellular and molecular mechanisms underlying the morphogenetic changes that participate in shaping the mature intestinal tract in vertebrates.
- PublicationOpen AccessImmunohistochemical evidence for an impairment of autophagy in tumorigenesis of gastric carcinoids and adenocarcinomas in rodent models and patients(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Vigen, Reidar Alexander; Kodama, Yosuke; Viset, Trond; Fossmark, Reidar; Waldum, Helge; Kidd, Mark; Wang, Timothy C.; Modlin, Irvin M.; Chen, Duan; Zhao, Chun-MeiBackground/Aim: Autophagy has dual roles in tumorigenesis: tumor-promoting or tumorsuppressing. The aim of the present study was to examine autophagy-related markers by immunohistochemistry in gastric carcinoids and adenocarcinomas in rodent models and patients. Methods: Gastric carcinoids in Mastomys were induced by loxtidine treatment. Spontaneously developed gastric adenocarcinomas in Japanese cotton rats and INS-GAS transgenic mice were included. Patient tissue samples of gastric carcinoids or adenocarcinomas were collected. Immunohistochemistry was performed against autophagy-related gene protein-6 (ATG-6, also called beclin-1), ATG-5 and ATG-16. Results: In tumor-free Mastomys, ATG-5, ATG-16 and beclin-1 were immunepositive in the gastric mucosa. In tumor-bearing Mastomys, ATG-5 and ATG-16 were negative in the tumors, whereas beclin-1 was positive in four of five animals. In carcinoid patients, ATG-5 was negative in six of ten, ATG-16 negative in nine of ten, and beclin-1 negative in three of ten patients. In cotton rats, ATG-5 and ATG-16 were negative in all tumors. Beclin-1 was negative in three of five rats. In INS-GAS mice, ATG-5 and beclin-1 were positive in the tumor area, but the numbers of immunopositive cells per gland were reduced by about 50% in comparison with wildtype mice. In adenocarcinoma patients, ATG-5 and ATG-16 were negative in eight of ten, and beclin-1 positive in all ten patients. Conclusions: An impaired autophagy took place at the stage of formation of ATG-5-ATG-12-ATG-16 complex in both gastric carcinoids and adenocarcinoma of both rodent models and patients. ATG-5 and ATG-16 might be better markers than beclin-1 in assessing autophagy in these lesions.
- PublicationOpen AccessElevated nuclear S100P expression is associated with poor survival in early breast cancer patients(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Maciejczyk, Adam; Łacko, Aleksandra; Ekiert, Marcin; Jagoda, Ewa; Wysocka, Teresa; Matkowski, Rafal; Hałoń, Agnieszka; Györffy, Balázs; Lage, Hermann; Surowiak, PawelS100P - low molecular weight acidic protein has been shown to be involved in processes of proliferation, survival, angiogenesis, multidrug resistance and metastasis in various human malignancies. In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumour behaviour, indicating that this protein may have adverse prognostic value. We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer patients with a median follow up of 17 years. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. We also studied prognostic value of S100P mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. Moreover, the relationship was examined between expression of S100P in cells of four breast cancer cell lines and their sensitivity to the 11 most frequently applied cytotoxic drugs. Univariate and multivariate analyses showed that higher expression of nuclear S100P (S100Pn) was typical for cases of a shorter overall survival and disease-free time. KM plotter analysis showed that elevated S100P expression was specific for cases of a relapse-free survival and distant metastases-free survival. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. We demonstrated that a high S100Pn expression level was associated with poor survival in early stage breast cancer patients. Since preliminary data indicated that expression of S100P was up-regulated by activation of glucocorticoid receptor and several agents manifested potential to activate or inhibit S100P promoter activity, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value.
- PublicationOpen AccessSerrated adenoma of the stomach: A clinicopathologic, immunohistochemical, and molecular study of nine cases(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Kwon, Mi Jung; Min, Byung-Hoon; Lee, Sun-Mi; Park, Ha Young; Kang, So Young; Ha, Sang Yun; Lee, Jun Haeng; Kim, Jae J.; Park, Cheol-Keun; Kim, Kyoung-MeeGastric serrated adenoma is a recently recognized entity that has been rarely described and poorly characterized. To examine whether gastric serrated adenoma shares the same immunophenotypic and molecular features of its colorectal traditional serrated adenoma, the clinicopathologic features, expression of mucin proteins (MUC2, MUC5AC, CD10, MUC6) and mismatch repair protein (MLH1), and mutations of BRAF and KRAS genes were studied. The nine serrated adenomas were obtained from five men and four women, with a mean age of 67 years. Seven (78%) serrated adenomas were located in the body of the stomach. The endoscopic findings were not sufficiently characteristic to diagnose serrated adenoma or serrated adenocarcinoma; however, most were elevated lesions. The initial biopsy material was available in all cases and the serrated features were evident in 6 cases diagnosed as adenoma. Among the nine cases, seven (78%) were associated with invasive adenocarcinoma within the serrated adenoma. MUC5AC was expressed in 6 serrated adenomas (67%). Expression of MUC5AC was observed in all tumors located in the lower third of the stomach. Focal MUC6 expression was observed in the basal part of two serrated adenomas. MLH1 expression was lost in two cases (22%). KRAS mutations were observed in three cases (33%) while BRAF mutations were not detected in any of the cases. Gastric serrated adenoma does not completely share the same immunophenotypic and molecular features of its colorectal counterpart. Gastric serrated adenomas are frequently associated with adenocarcinoma. When serrated adenoma is encountered in a gastric biopsy specimen, the possibility of associated adenocarcinoma should be considered in the adjacent stomach.
- PublicationOpen AccessThe origin of lipofuscin in brown adipocytes of hyperinsulinaemic rats: the role of lipid peroxidation and iron(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Markelic, Milica; Velickovic, Ksenija; Golic, Igor; Klepal, Waltraud; Otasevic, Vesna; Stancic, Ana; Jankovic, Aleksandra; Vucetic, Milica; Buzadzic, Biljana; Korac, Bato; Korac, AleksandraThe aim of this study was to investigate lipofuscin origin in brown adipocytes of hyperinsulinaemic rats and the possible role of lipid peroxidation and iron in this process. Ultrastructural examination revealed hyperinsulinaemia-induced enhancement in the lipofuscin production, accompanied by an increase of mitochondrial damage in brown adipocytes. Extensive fusions of lipid droplets and mitochondria with lysosomes were also observed. Confocal microscopy showed lipofuscin autofluorescence emission in brown adipose tissue (BAT) after excitation at 488 nm and 633 nm, particularly in the insulin-treated groups. The presence and distribution of lipid peroxidation product, 4-hydroxy-2-nonenal (4- HNE), in brown adipocytes was assessed by immunohistochemical examination revealing its higher content after treatment with insulin. The iron content was quantified by electron dispersive X-ray analysis (EDX) showing its higher content in the hyperinsulinaemic groups. The ultrastucture of the majority of lipofuscin granules suggests their mitochondrial origin, which was additionally confirmed by their colocalization with ATP synthase. In conclusion, our results suggest that increased lipofuscinogenesis in the brown adipocytes of hyperinsulinaemic rats is a consequence of lipid peroxidation, mitochondrial damage and iron accumulation.
- PublicationOpen AccessLack of cell stress markers in fibrous cap cells in the left main coronary artery(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Rai, Sudarshan; Zulli, AnthonyFibrous cap formation is a key aspect of preventing clinical events but animal models to study this are limited and cellular stress plays a fundamental role in fibrous cap formation. Aims: To characterise cellular stress markers in an established animal model to study coronary artery fibrous cap formation. Methods: Male New Zealand White rabbits were fed a diet containing 0.5% cholesterol and 1% methionine for 4 weeks, then 9 weeks of normal diet to induce fibrous cap formation. Immunohistochemistry was used to detect CHOP, GRP78, nitrotyrosine HSP70, HSP90, iNOS and HSP32. Results: The core within the left main coronary artery atherosclerosis contained vast amount of foamy macrophages which readily stained for all markers. However, the smooth muscle cells within the formed fibrous cap were negative for all markers. The endothelium overlying the fibrous cap was positive for CHOP, GRP78, nitrotyrosine, iNOS and HSP32, however it was difficult to detect positive endothelial HSP70 or HSP90 immunoreactivity. Serial sectioning and immunohistochemistry for all factors showed clear dual iNOS+ / HSP32+ / HSP70- / HSP90- single cells within the fibrous cap formed. Conclusion: Smooth muscle cells within fibrous caps appear ‘stress free’, however isolated single smooth muscle cells within caps and within the core show positive immunoreactivity for stress markers. This model could be used to understand the role of cellular stress in fibrous cap formation in the coronary artery.