Histology and histopathology Vol.24, nº4 (2009)
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- PublicationOpen AccessImmunohistochemical expression of glycodelin in breast cancer correlates with estrogen-receptor α and progesterone-receptor A positivity(Murcia : F. Hernández, 2009) Scholz, Christoph; Toth, Bettina; Barthell, Elisabeth; Mylonas, I.; Weissenbacher, Tobias; Friese, Klaus; Jeschke, UdoGlycodelin (Gd), previously known as placental protein 14 (PP 14), acts as an immunosuppressive glycoprotein by suppressing the cytolytic capacity of human natural killer (NK) cells and T-cells in vitro. Glycodelin is expressed in normal glandular epithelium of the endometrium as well as in normal and malignant glandular cells in and outside of the reproductive tract. Recently, Gd expression was demonstrated in normal and cancerous human breast tissue. Paraffin-embedded breast cancer tissue blocks (n=121) were examined for Gd expression. No part of the specimens contained carcinoma in situ. Gd expression was present in lobular and ductal breast carcinoma. We observed expression of Gd in breast cancer independent of grading. With regard to nodal status, no significant differences in the expression of Gd between cancer tissue from patients with or without axillary lymph node metastases were present. However, Gd expression was found to be significantly higher in breast cancer tissue when the staining reaction for steroid receptors was also positive. These results implicate that Gd might be an additional marker for the differentiation of breast cancer tissue. To which extent Gd could serve as an additional indicator for breast cancer survival is part of our ongoing research.
- PublicationOpen AccessBiomarkers for novel targeted therapies of hepatocellular carcinoma(Murcia : F. Hernández, 2009) Okamoto, Kinya; Neureiter, DanielIncreasing insights into molecular alterations of signalling pathways have led to the development of specific targeted therapies for cancer. Due to the high specificity of monoclonal antibodies or small molecule inhibitors, identification of patients who will benefit from these therapeutics is crucial for treatment success. Furthermore, as classical endpoints of clinical trials are not fully applicable to targeted therapies, biomarkers for monitoring treatment response have to be identified. The recent introduction of a multi-kinase inhibitor for the treatment of liver cancer has accelerated efforts in the field of biomarker research. As further novel targeted therapies are on the horizon for liver cancer therapy, we will here review candidate markers for new hepatocellular carcinoma therapies, with a focus on EGF- and VEGF-receptor related pathways.
- PublicationOpen AccessLiver growth factor antifibrotic activity in vivo is associated with a decrease in activation of hepatic stellate cells(Murcia : F. Hernández, 2009) Díaz-Gil, Juan J.; García-Monzón, Carmelo; Rúa, Carmen; Martín Sanz, Paloma; Cereceda, Rosa M.; Miquilena-Colina, María E.; Machín, Celia; Fernández Martínez, Amalia; García Cañero, RafaelThe antifibrotic activity of Liver Growth Factor (LGF), a liver mitogen, was previously demonstrated in several models of rat liver fibrosis and even in extrahepatic sites, such as carotid artery in hypertensive rats and rat CdCl2-induced lung fibrosis. In the present study, we have attempted to examine in depth its mechanism of antifibrotic action in bile duct-ligated (BDL) rats, with special emphasis on its activity in fibrogenic liver cells. BDL rats received either LGF 9 μg/week for 2 or 3 weeks (BDL+LGF, n=20/group) or saline (BDL+S, n=20/group), at times 0, week 2, or week 5 after operation. Groups were compared in terms of liver a- smooth muscle actin (SMA) content (western blotting and immunohistochemistry), hepatic apoptosis, liver desmin content (western blotting), and serum endothelin-1 (ELISA). LGF produced a marked decrease in liver a-SMA content compared with saline-injected rats, especially evident at longer times (5w and 8w; p<0.05), accompanied by a decrease in hepatic a-SMA+ cells. This decrease was not due to the killing of activated hepatic stellate cells (HSC) or myofibroblasts by LGF, since there was a slight decrease in hepatic apoptosis that was more evident at 2w (p<0.05). Moreover, LGF did not seem to influence HSC proliferation, as shown by measuring liver desmin content. The antifibrotic activity exerted by LGF seems to be closely related to a modulation of the activation state of fibrogenic liver cells (activated HSC and myofibroblasts) in BDL rats.
- PublicationOpen AccessMGMT analysis at DNA, RNA and protein levels in glioblastoma tissue(Murcia : F. Hernández, 2009) Preusser, MatthiasEvidence from several studies supports that the epigenetic, transcriptional and translational regulation and expression of O6-methylguaninemethyltransferase (MGMT) is relevant for prognostic and predictive considerations in glioblastoma patients. MGMT status is being used as a stratifying factor or eligibility criterion in ongoing and accruing clinical glioblastoma trials. In some cases, there is also interest in MGMT assessment of glioblastoma tissue in the dayto- day clinical setting. However, a number of different methods and protocols have been used for MGMT analysis and it is unclear which methods harbour the greatest potential for translation into routine clinical use. This article reviews methods that have been used for MGMT assessment at DNA-, RNA- and protein-level in glioblastoma with a focus on their potential clinical utility. Conclusions. (1) DNA-based methods for MGMT analysis seem more promising for translation into the clinical setting than RNA- or protein-based methods. However, at present there is lack of data to base recommendations for a specific method or protocol for MGMT testing on. There is a strong need for systematic comparisons and validation of intra- and interlaboratory reproducibility and clinical performance of different methods for MGMT assessment to identify the best method for clinical MGMT testing. (2) The current practice of formalin-fixation of neurosurgical specimens considerably limits the spectrum of methods that can be applied for molecular diagnosis in clinical neurooncology. Further studies may be helpful to establish more appropriate protocols for tumour tissue preservation (e.g. identification of alternative fixatives that do not deteriorate DNA and RNA quality).
- PublicationOpen AccessThe interactomics of sortilin: an ancient lysosomal receptor evolving new functions(Murcia : F. Hernández, 2009) Canuel, Maryssa; Libin, Yuan; Morales, Carlos R.The delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6- phosphate receptor (MPR). The MPR has been demonstrated to attain the early endosomes via a process that requires the interaction of its cytosolic domain with the GGA and AP-1 adaptor proteins. Additionally, the MPR can be recycled back to the trans-Golgi network (TGN) through its interaction with the retromer complex. Interestingly, in I-cell disease (ICD), in which the MPR pathway is non-functional, many soluble lysosomal proteins continue to traffic to the lysosomes. This observation led to the discovery that sortilin is responsible for the MPR-independent targeting of the sphingolipid activator proteins (SAPs) and acid sphingomyelinase (ASM). More recently, our laboratory has tested the hypothesis that sortilin is also capable of sorting a variety of cathepsins that exhibit varying degrees of MPR-independent transport. We have demonstrated that the transport of cathepsin D is partially dependent upon sortilin, that cathepsin H requires sortilin, and that cathepsins K and L attain the lysosomes in a sortilin-independent fashion. As a type-1 receptor, sortilin also has numerous cytosolic binding partners. It has been observed that like the MPR, the anterograde trafficking of sortilin and its cargo require both GGAs and AP-1. Similarly, the retrograde recycling pathway of sortilin also involves an interaction with retromer through a YXXf site in the cytosolic tail of sortilin. In conclusion, the cytosolic domains of sortilin and MPR possess a high degree of functional homology and both receptors share a conserved trafficking mechanism.
- PublicationOpen AccessLocalization of the polyol pathway in the human kidney(Murcia : F. Hernández, 2009) Zopf, Steffen; Flämig, Jakob; Schmid, Heide; Miosge, Nicolai; Blaschke, Sabine; Hahn, Eckhart G.; Múller, Gerhard Anton; Grunewald, Rolf W.Sorbitol plays an important role in the osmotic regulation of the mammalian kidney. Sorbitol synthesis is regulated by the enzyme aldose reductase (AR) and its degradation to fructose is catalyzed by the enzyme sorbitol dehydrogenase (SDH). Various data exist on the polyol pathway on the rat kidney, but little is known about the distribution of the polyol pathway enzymes in the human kidney. Determination of enzyme activities and a semiquantitative determination of mRNA expression, immunohistochemistry and in-situ hybridisation in healthy human kidney tissue was carried out. The enzyme activity of AR showed a fourfold increase from cortex to papilla, while SDH-activity dropped from cortex to papilla by a factor of four. Corresponding data was obtained at the mRNA level from the semiquantitative polymerase chain reaction (PCR). Additional differentiation at the cellular level reveals both enzymes in cells of the proximal and distal tubules, thick ascending loop, thin loop and collecting duct. Studies of enzyme activity and expression by immunohistochemistry, PCR and in-situ hybridization presented corresponding results with respect to the localization of the enzymes, which match the experimental data obtained from rats very well. Thus, the established rat model might well represent the situation in the human kidney, too.
- PublicationOpen AccessAnalysis of the molecular expression profile of non small cell lung carcinoma associated to chronic obstructive pulmonary disease(Murcia : F. Hernández, 2009) Garcia-Lujan, Ricardo; Conde-Gallego, Esther; Lopez-Ríos, Fernando; Martin de Nicolas, Jose Luis; Sanchez-Céspedes, Montserrat; García-Quero, Cristina; Echave-Sustaeta, José María; Lopez-Encuentra, AngelChronic obstructive pulmonary disease (COPD) is an independent risk factor to develop lung cancer but there are no different functional clusters of biomarkers between patients with non-small cell lung cancer (NSCLC) with or without COPD. To analyse protein expression, in order to find out whether samples of resected NSCLC from patients with COPD present a different molecular expression. Observational, cohort, concurrent study with sampling since treatment of disease in patients with NSCLC in initial stages (pIApIIB) treated surgically in our hospital between October 1993 and September 1997. The study consisted of the elaboration of tissue arrays with samples from resected tumor, using immunohistochemistry as a study method. Univariate analysis and logistic regression analysis were performed in order to determine molecular markers that showed a differential expression in NSCLC of the patients with COPD. We studied thirty-two proteins in 146 patients. 30% of the patients had COPD. Univariate analysis in patients with COPD showed one molecular marker to be overexpressed and five molecular markers to be underexpressed. Multivariate analysis in patients with COPD identified membranous ß-Catenin as a differential biomarker, which displayed an underexpression, with an Odds Ratio (95% Confidence Interval) of 0.26 (0.07-1.01). A significant lowest expression of membranous ß-catenin was detected in NSCLC of the patients with COPD.
- PublicationOpen AccessDensity and migration of mast cells in lip squamous cell carcinoma and actinic cheilitis(Murcia : F. Hernández, 2009) Lago Costa, Nádia; Ferreira Oton-Leite1, Angélica; Peixoto Cheim-Júnior, Adonai; Gonçalves Alencar, Rita de Cássia; Jabur Bitta, Glória Oton; Aparecida Silva, Tarcília; Carvalho Batista, AlineMast cells (MCs) display a diversity of roles that may contribute to the stromal microenvironment alterations during tumor progression. The aim of this study was to investigate MC populations expressing tryptase and c-kit in lip squamous cell carcinoma (lip SCC) (n=37), actinic cheilitis (AC) (n=15) and normal lip mucosa (control) (n=6), as well as their relationship with microscopic parameters (collagen degeneration, elastin changes, angiogenesis and proliferative index). Tryptase, c-kit, CD31 and Ki-67 expressions were analyzed by means of immunohistochemistry and collagen and elastic fibers were visualized with Picrosirus and Verhoeff’s stain, respectively. The numbers of tryptase+ MC were significantly higher in lip SCC when compared with control (P=0.01), while a similar density of these cells was observed in AC and lip SCC (P=0.09). The density of c-kit+ MC was similar in all groups examined (P=0.65). MC migration (ckit+/Tryptase+ relationship) was 69% in lip SCC, 60% in AC and 100% in control. The number of CD31+ blood vessels was significantly higher in the lip SCC when compared with control and AC (P<0.01). The increase of MCs and angiogenesis in lip SCC may reflect an important modification in the tumor microenvironment during squamous photocarcinogenesis.
- PublicationOpen AccessInvestigating the ultrastructure of platelets of HIV patients treated with the immuno-regulator, Canova: a qualitative scanning electron microscopy study(Murcia : F. Hernández, 2009) Pretorius, E.; Smit, E; Oberholzer, H.M.; Briedenhann, S.; Franz, R.C.; Steyn, E.The resistance of HIV strains to the available antiretroviral medication has become a major problem in the world today. This has forced researchers to investigate the possible use of alternative drugs such as homeopathic medicine (e.g. immunomodulators) to enhance the immune system of patients infected with HIV. Canova is an immunomodulator of herbal origin which is known to stimulate the host defense against several pathological states through the activation of the immune system. Blood platelets play an important role in homeostasis, thrombosis and the immune response by forming platelet aggregates. The ultrastructure of platelet aggregates of patients with HIV has been studied previously using SEM to determine the effect of HIV on the platelet morphology. Membrane blebbing and ruptured platelet membranes were observed which is indicative of apoptosis, revealing that HIV patients may develop thrombocytopenia as a result of peripheral platelet destruction. The aim of the current study was to investigate the effect of HIV on the morphology of platelets from patients treated with the immunomodulator, Canova, compared to control individuals and HIV patients not on the Canova treatment. Blood was drawn from the individuals and the coagula were formed by adding human thrombin to the platelet rich plasma. Examination was done using SEM. CD4 counts were also determined. Slight morphological changes were seen when comparing the fibrin networks from the control, untreated HIV patients and the Canova-treated HIV patients, suggesting that HIV does not impact on the fragility of fibrin networks. In HIV patients there are bleb-like bulges on the membrane of platelets as well as membrane breakages visible on the aggregate, whereas in the Canova-treated patients membrane blebbing is far less pronounced and there are large areas of intact, smooth membranes with visible canalicular areas, suggesting that Canova protects the membranes of platelets and that blebbing does not appear in such great proportions as was found in the untreated HIV group. These results support and provide ultrastructural evidence for the results seen in previous research, where it is seen that Canova protects the immune system of immuno-compromised patients by keeping the ultrastructure intact thereby preventing the devastating cyto-destructive effects of HIV disease.
- PublicationOpen AccessPrognostic significance of tumour vascularisation on survival of patients with advanced ovarian carcinoma(Murcia : F. Hernández, 2009) Labiche, Alexandre; Elie, Nicolas; Herlin, Paulette; Denoux, Yves; Crouet, Hubert; Heutte, Natacha; Joly, Florence; Héron, Jean-François; Gauduchon, Pascal; Henry-Amar, MichelObjective. The prognostic significance of microvessel density in ovarian cancer is still a matter of debate. Classically, the degree of vascularisation is assessed in areas of high vascular density (hot spots), considered as regions of increased probability of metastasis. Since ovarian tumours have a particular progression and dissemination behaviour, vascularisation outside hot spots may also contribute to their evolution. Methods. In the present study, the degree of tumour vascularisation was estimated both in whole histogical sections and in hot spots, in 235 patients with ovarian carcinoma, using fully automatic image analysis methods. Six parameters were estimated: mean microvessel density (MVD) and mean microvessel surface fraction (MSP) on the whole section, mean and maximum values of MVD and MSP inside hot spots (MVDHS1, MSPHS1 and MVDHS2, MSPHS2). Relationships between vascular parameters and clinicopathologic features were analysed. Results. In stage III-IV patients multivariate analysis showed that stage IV disease (hazards ratio (HR)=1.72, p=0.001), post-surgical residual disease 1cm (HR=2.86, p<0.001), upper MVD tercile (HR=1.45, p<0.022) and medial MVDHS1 tercile (HR=1.36, p=0.060) retained an independent prognostic value upon overall survival. Conclusion. Our results suggest that quantification of blood vessels, both on the whole histological section and in hot spots might be helpful in evaluating prognosis in advanced ovarian carcinomas.
- PublicationOpen AccessFocal adhesion kinase and cancer(Murcia : F. Hernández, 2009) Golubovskaya1, Vita M.; Kweh, Frederick A.; Cance, William G.Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that resides at the sites of integrin clustering, known as focal adhesions. The FAK protein has a molecular mass of 125kDa and is encoded by the FAK gene located on human chromosome 8q24. Structurally, FAK consists of an amino-terminal regulatory FERM domain, a central catalytic kinase domain, two proline-rich motifs, and a carboxy-terminal focal adhesion targeting domain. FAK has been shown to be an important mediator of cell growth, cell proliferation, cell survival and cell migration, all of which are often dysfunctional in cancer cells. Our lab was the first to isolate FAK from primary human tissue and link it to the process of tumorigenesis. We analyzed FAK mRNA expression in normal, invasive and metastatic human tissues and demonstrated through Northern blot analysis that normal tissues had very low levels of FAK mRNA while primary and metastatic tumors significantly overexpressed FAK. We also demonstrated and confirmed FAK overexpression in colorectal carcinoma and liver metastases with real-time PCR. In this review we summarized immunohistochemical data of FAK expression and role in different cancer types tumors and discussed FAK inhibition therapy approaches.
- PublicationOpen AccessPineal concretions in turkey (Meleagris gallopavo) as a result of collagen-mediated calcification(Murcia : F. Hernández, 2009) Przybylska-Gornowicz, B.; Lewczuk, B.; Prusik, M.; Bulc, M.The intra-pineal calcification is a well-known phenomenon in mammals, however it is almost completely unknown in birds. The aim of the present work was to analyze morphology and genesis of the pineal concretions in the turkey. The studies were performed on the pineals collected from one-year-old turkeys (Meleagris gallopavo). In addition to standard morphological methods, the alizarin red S and potassium pyroantimonate methods were employed for localization of calcium at the light and electron microscopy level. In light microscopy, calcified concretions with diameters from 300 μm to 2 mm and quantities from 3 to 6 per gland were observed in all the examined pineals. They were stained red with alizarin S and showed the presence of collagen in Mallory's staining. Two types of cells were noted inside the concretion: polygonal and elongated ones. Using electron microscopy, three parts were distinguished within the calcification area. The peripheral part contained densely packed collagen fibrils, some elongated cells and numerous pyroantimonate precipitates demonstrating the presence of calcium ions. In the intermediate part, the fibrils were covered by almost continuous sheets of pyroantimonate precipitates and fused side by side. The central part showed an appearance of calcified hard tissue and showed an appearance of calcified hard tissue and contained some polygonal (osteocyte-like) cells. The obtained data demonstrated that the formation of the pineal concretions in the turkey is associated with the mineralization of collagen. This process is completely different from the mechanisms responsible for the formation of the concretions in the mammalian pineal.
- PublicationOpen AccessActivation of matrix metalloproteinase (MMP)-2 by membrane type 1-MMP and abnormal immunolocalization of the basement membrane components laminin and type IV collagen in canine spontaneous hemangiosarcomas(Murcia : F. Hernández, 2009) Murakami, M.; Sakai, Hidetaka; Kodama, A.; Mori, T.; Yanai, T.; Maruo, K.; Masegi, T.We performed immunohistochemical investigation of the basement membrane (BM) components, namely, type IV collagen and laminin, in 83 canine hemangiosarcomas (HSAs), 22 hemangiomas, and some granulation tissues (GTs). Additionally, we analyzed the expression and activities of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP (MT1-MMP) using the same samples by immunohistochemistry and gelatin zymography to investigate whether MMPs were associated with the BM degradation. In immunohistochemistry for the BM components, many HSAs showed discontinuous linear/negative immunoreactivity in the BM (type IV collagen: 49.4%/14.5%, laminin: 60.3%/10.8%, respectively). In contrast, almost all hemangiomas showed continuous staining in the BM (type IV collagen: 90.9%, laminin: 95.5%, respectively). Interestingly, positive cytoplasmic immunoreactivity for type IV collagen and laminin was observed in 97.6% and 91.6% HSA, respectively. Although MMP-9 immunoreactivity wasn’t detected in neoplastic and active angiogenic endothelial cells (ECs), MMP-2 was detected in all ECs of GTs and in neoplastic cells of both vascular tumors. A strong immunoreactivity for MT1- MMP was observed in active angiogenic ECs in GTs and in neoplastic ECs in HSAs. However, almost all hemangiomas showed weak/negative immunoreactivity. In gelatin zymography, significantly strong activity of active MMP-2 was observed in HSAs, similar to that in active angiogenesis in GTs; however, weak/no activity of active MMP-2 was detected in hemangiomas. In canine HSA, neoplastic cells had active MMP-2, possibly activated by MT1-MMP, and discontinuous status of BM might be associated with activity of active MMP-2.
- PublicationOpen AccessKAI-1CD82, The molecule and clinical implication in cancer and cancer metastasis(Murcia : F. Hernández, 2013-09-24) Malik1, Fraz Arshad; Sanders, Andrew J.; Jiang, W.G.-