Histology and histopathology Vol.24, nº7 (2009)
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- PublicationOpen AccessClaudin-5 protein is a new differential marker for histopathological differential diagnosis of canine hemangiosarcoma(Murcia : F. Hernández, 2009) Jakab, Cs.; Halász, J.; Kiss, A.; Schaff, Z.; Rusvai, M.; Gálfi, P.; Abonyi, T.Z.; Kulka, J.Aims: Claudin-5 protein is an endothelspecific claudin, present in tight junctions. To evaluate its usefulness as a differential diagnostic marker of canine hemangiosarcomas, the expression of claudin-5 molecule was studied in different canine tumours of vascular origin. Methods and results: Ninety two canine neoplastic tissue samples obtained from necropsies and biopsy specimens were routinely processed and stained immunhistochemically for claudin-5. The neoplastic endothelial cells of canine hemangiosarcomas, hemangiomas, and lymphangiomas showed a strong membrane immunoreactivity for claudin-5, but the other investigated canine malignant and benign tumours, including fibrosarcomas, myxo-, leiomyo-, cardiac rhabdomyo-, neurofibro-, synovial-, osteo-, and chondrosarcomas, spindle cell melanomas, hemangiopericytomas, benign fibroblast proliferations, and leiomyomas were negative for this endothelial marker. In these non-vascular canine tumours intense immunostaining was detected in the endothelial cells of the incorporated intratumoural vessels and neovasculature. The canine splenic hematomas induced by hemangiosarcomas were distinguished from splenic hematomas induced by non-neoplastic lesions by the means of claudin-5 protein. In hemangiosarcomas the percentage of positive neoplastic endothelial cells was higher, and stronger when using the claudin-5 molecule compared to CD31 and vWf. Conclusion: The results show that claudin-5 molecule can be used as a new differential marker, and could also be of a diagnostic value in the differential diagnosis of canine hemangiosarcomas from sarcomas of other origin with hemorrhages or increased vascularization. Claudin-5 could help to reveal neoplastic proliferation of endothelial cells causing splenic hematomas and differentiate these tumours from non-vascular neoplastic splenic lesion. The immunohistochemical detection of the claudin-5 protein had a higher sensitivity than CD31, and vWf antigen in case of canine hemangiosarcomas.
- PublicationOpen AccessImmunohistopathological and neuroimaging characterization of murine orthotopic xenograft models of glioblastoma multiforme recapitulating the most salient features of human disease(Murcia : F. Hernández, 2009) Radaelli, E.; Ceruti, R.; Patton, V.; Russo, M.; Degrassi, A.; Croci, V.; Caprera, F.; Stortini, G.; Scanziani, E.; Pesenti, E.; Alzani, R.Tumorigenesis in human glioblastoma multiforme (GBM) is driven by several genetic abnormalities with disruption of important molecular pathways, such as p53/MDM2/p14ARF and EGFR/PTEN/Akt/mTOR. The malignant progression of human GBM is also primarily associated with a peculiar multistep pathophysiological process characterized by intratumoral ischemic necrosis (i.e. pseudopalisading necrosis) and activation of the hypoxia-inducible factor (HIF)-1a pathway with consequent peritumoral microvascular proliferation and infiltrative behaviour. Predictable preclinical animal models of GBM should recapitulate the main pathobiological hallmarks of the human disease. In this study we describe two murine orthotopic xenograft models using U87MG and U251 human cell lines. Ten Balb/c nude male mice were orthotopically implanted with either U87MG (5 mice) or U251 (5 mice) cell lines. Intracranial tumor growth was monitored through Magnetic Resonance Imaging (MRI). Immunohistopathological examination of the whole cranium was performed 30 days after implantation. U251 orthotopic xenografts recapitulated the salient pathobiological features described for human GBM, including invasive behaviour, wide areas of pseudopalisading necrosis, florid peripheral angiogenesis, GFAP and vimentin expression, nonfunctional p53 expression, striking active-caspase-3 and HIF-1a expression along pseudopalisades. U87MG orthotopic xenografts proved to be very dissimilar from human GBM, showing expansile growth, occasional necrotic foci without pseudopalisades, intratumoral lacunar pattern of angiogenesis, lack of GFAP expression, fuctional p53 expression and inconsistent HIF-1a expression. Expression of pAkt was upregulated in both models. The results obtained suggest that the U251 orthotopic model may be proposed as a predictive and reliable tool in preclinical studies since it recapitulates the most salient pathobiological features reported for human GBM.
- PublicationOpen AccessExpression of beta-catenin and its mechanism of delocalization in intestinal-type early gastric cancer based on mucin expression(Murcia : F. Hernández, 2009) Lee, Soo Han; Kang, Hyun Jeong; Shin, Dong-Hun; Cho, Duk-Yeon; Song, Jin Mi; Kim, G.H.; Song, G.A.; So, Mee Young; Kim, J.Y.; Choi, K.U.; Lee, Chian-Her; Huh, G.Y.; Park, D.Y.; Lee, H.C.The biological characteristics of intestinaltype early gastric cancers (ICs) differ based on mucin phenotypes. Beta-catenin delocalization is a predictive marker of aggressive biological behavior (submucosal invasion and lymph node metastasis) of ICs. The presumptive causative genetic alterations leading to delocalization of beta-catenin in ICs are still controversial, and there are only a few reports regarding beta-catenin expression in gastric cancer based on mucin phenotypes. Therefore, in the current study, the expression and mechanisms of delocalization of betacatenin were elucidated on the basis of mucin phenotypes in 109 cases of ICs. There was increased cytoplasmic and nuclear beta-catenin expression (delocalization) in ICs with a predominant intestinal mucin phenotype (ICIP; 46.3% [25/54 cases]) compared to ICs with a predominant gastric mucin phenotype (ICGP; 20% [11/55 cases]). There were no beta-catenin or APC mutations in ICs. APC promoter hypermethylation was present in 49 of 105 (46.7%) cases of ICs. There was a significant relationship between APC promoter hypermethylation and betacatenin delocalization in ICs, especially in ICIPs. There was no relationship between beta-catenin delocalization and APC gene loss of heterozygosity in ICs. In conclusion, we showed that beta-catenin delocalization was more evident in ICIPs, and APC promoter hypermethylation might play a role in delocalization of beta-catenin, especially in ICIPs.
- PublicationOpen AccessAnalysis of gene status in cervical dysplastic lesions and squamous cell carcinoma using tissue microarrays(Murcia : F. Hernández, 2009) Costa, C.; Espinet, Blanca; Molina, Miguel A.; Salgado, Rocio; Salido, M.; Baró, Teresa; Fusté, P.; Mancebo, G.; Carreras, R.; Solé, Francesc; Serrano, S.; Alameda, F.Cervical displasia are classified as CIN-I, CIN-II and CIN-III. It has been observed that in at least 60% of CIN-I and CIN-II, the pathology disappears spontaneously, while around 30% persist at 24 months, 10% progress to CIN-III and 1% develops as a SCC. The factors involved in the evolution of the pathology are not defined, although infection of HPV is a necessary condition, but not the only one. For this reason, the identification of genetic changes is an essential element for understanding the carcinogenic process. It can also serve as a helpful tool for identifying patients who may be susceptible to its evolution and treatment, from patients whose lesions could regress spontaneous and for whom periodic follow-ups would be enough. Fiftty three cervical biopsies from patients with dysplasia and ISCC were included in the study. These biopsies were set into nine macroarrays. Eight genes and five proteins were examined in each samples (hTERT, PIK3CA, hTERC, MYC, CCND1, BCL2, ZNF217 and p16) by fluorescence in situ hybridization (FISH) and/or immunohistochemistry (IHC). The results reflected that the genetic alterations of PIK3CA, ZNF217 and CCND1 were associated with the evolution of normal tissue to CIN I, those of hTERC and ERBB with the evolution of LSIL to HSIL, those of hTERT and MYC with the evolution of CIN-II/CIN-III to ISCC, and those of BCL-2 with the inception of ISCC. With regards to proteins, the expression of MYC and CCND1 in the initial stages of the illness would help in the acquisition of the altered cellular phenotype.
- PublicationOpen AccessTissue distribution of perlecan domains III and V during embryonic and fetal human development(Murcia : F. Hernández, 2009) Roediger, Matthias; Kruegel, Jenny; Miosge, Nicolai; Gersdorff, NikolauA major component of basement membranes (BMs) is perlecan, a five-domain heparan sulphate proteoglycan. During murine embryogenesis, nearly all BMs of mesenchymal origin express perlecan, and it is believed to participate in the supramolecular assembly of BMs. However, the distribution of perlecan in human embryonic and fetal tissues is widely unknown, except for cartilage anlagen of developing extremities and the fetal spine. Clinical syndromes, caused by perlecanassociated mutations or gene-defects, suggest its multifunctional involvement during human development. Here we reveal the immunohistochemistry of perlecan domains III and V during human development from gestational weeks (gw) 6 to 12 in basement membrane zones (BMZs) of the developing brain, nervous system, blood vessels, skin, lung, heart, kidney, liver, intestine and skeletal system. Interestingly, a difference in the distribution of the two perlecan domains was found in the endoneurium of ganglia. Domain III is strongly present from gw 6 onwards, while domain V shows attenuated expression at this stage and has been detected abundantly only from gw 8 onwards, possibly indicating vascularization of the endoneurium during this early stage. We found perlecan to be present particularly at those stages of human development where epithelial-mesenchymal interactions occur.
- PublicationOpen AccessTumor stroma is the predominant uPA-, uPAR-, PAI-1-expressing tissue in human breast cancer: prognostic impact(Murcia : F. Hernández, 2009) Hildenbrand, Ralf; Schaaf, Antonela; Dorn-Beineke, Alexandra; Allgayer, Heike; Sütterlin, Marc; Marx, Alexander; Stroebel, PhilippUrokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor PAI-1, play a key role in tumor invasion and metastasis. uPA and PAI-1 were the first novel tumor biological factors to be validated at the highest level of evidence regarding their clinical utility in breast cancer. Their antigens are determined in tumor tissue extracts by standardized, quality-assured immunometric assays (ELISA). Since the late 1980s, numerous independent studies have demonstrated that patients with low levels of uPA- and PAI-1 in their primary tumor tissue have significantly better survival than patients with high levels of either factor. However, it is unclear whether it is their (relative) levels in the tumor stroma or in the tumor cells themselves that is most relevant to patient outcome. This missing knowledge leads to an uncertainty concerning the management of breast cancer tissue specimens. It is unclear how much tumor stroma is allowed in one tumor tissue specimen for an adequate assessment of the patients' outcome. This is the first study in which tumor cells and stromal tissue of invasive breast carcinomas (n=60) were separated by laser capture microdissection followed by ELISA-based determination of the uPA-, uPAR- and PAI-1-levels. In addition, we have assessed uPA-, uPAR- and PAI-1 distribution in formalin-fixed, paraffin-embedded breast cancer specimens (n=60) by immunohistochemistry. The uPA-, uPAR- and PAI-1 in tumor stroma only, tumor cells only and not separated tumor tissue did not show any significant differences in protein-levels determined by ELISA. Cox regression analysis showed that patients with high uPA-, high uPAR-, and/or high PAI-1-levels, as compared to patients with low levels of either factor, showed a significantly shorter relapse-free survival and overall survival (p=0.000001). These results suggest that a strong expression of uPA, uPAR and PAI-1 in the tumor stroma, as well as in tumor cells, have the same impact on the clinical behaviour of breast cancer. Conclusion: When using uPA- and PAI-1 levels as prognostic and predictive factors in breast cancer the quantity of tumor stroma in the tumor tissue specimen is not relevant for the assessment of the patients' outcome.
- PublicationOpen AccessKeratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis(Murcia : F. Hernández, 2009) Moretti, Silvia; Fabbri, Paolo; Baroni, Gianna; Berti, Samantha; Ban, Daniele; Berti, Emilio; Nassini, Romina; Lotti, Torello; Massi, DanielaVitiligo is a skin disorder characterized by loss of functional melanocytes. Keratinocytes contribute to melanocyte homeostasis, and keratinocyte alteration may play a role in melanocyte dysfunction in vitiligo. In particular, the release of melanogenic mediators and the level of functioning keratinocytes may affect melanocyte dysfunction in vitiligo epidermis. Keratinocyte-derived mediators involved in pigmentation, analysed by in situ hybridization, and epidermal apoptosis, detected by TUNEL assay and electron microscopy, were evaluated in lesional and perilesional skin biopsies from 15 patients with active vitiligo and in 5 control subjects. Among the melanogenic mediators, stem cell factor (SCF) and endothelin-1 (ET-1) mRNA were significantly reduced in lesional as compared to perilesional epidermis, whereas no difference was observed in mRNA of basic fibroblastic growth factor (bFGF) and granulocyte-monocyte colony stimulating factor (GM-CSF). The expression of mRNA for tumor necrosis factor (TNF)-α and interleukin-6 (IL-6), two pro-inflammatory cytokines with an inhibitory effect on pigmentation, was increased in the epidermis from vitiligo biopsies, whereas their expression was practically undetectable in the skin of control subjects. Apoptotic keratinocytes were more abundant in lesional vs. perilesional skin of vitiligo patients and were absent in the epidermis of control subjects. Changes in expression of keratinocyte-derived mediators observed in the present study are consistent with their differential functions in melanocyte regulation. In particular, increased TNF-α could contribute to keratinocyte apoptosis, which results in reduced release of melanogenic cytokines and ultimately in melanocyte disappearance.
- PublicationOpen AccessThe pathogenesis of chronic inflammation and malignant transformation in the human upper airways, the role of beta-defensins, eNOS, cell proliferation and apoptosis(Murcia : F. Hernández, 2009) Pacova, H.; Astl, J.; Martinek, J.The surrounding environment contains plenty of pathogens, which represent a danger of infection. The simplest way for the pathological microorganism to enter the organism is the upper airways. Inflammation of the upper airways is among the most common and frequent diseases. This category includes nasal polyposis and chronic tonsillitis. In many cases it is associated with disorders in relation to the immune response. An inflammatory infiltration of mononuclears, eosinophils, plasma and mast cells can be found in the histological structure of the polypous as well as tonsillar mucosa. One aim of this study was to determine the expression of beta-defensins and various proteins, with a possible potential role in relation to the rise and development of those changes. Another aim was to determine the relationship between the inflammatory and malignant processes in the tonsils. The samples of nasal polyps were obtained during clinically indicated endonasal surgery from patients diagnosed with nasal polyposis (n=50). The samples of tonsils were collected during surgery from patients suffering from chronic tonsillitis (n=11) or tonsillar carcinoma (n=17). Immunohistochemical procedures for the detection of human beta-defensin 1, 2, 3 (HBD-1, 2, 3), Ki- 67, endothelial nitric oxide synthase (eNOS) and cleaved caspase 3 were performed on cryostate and paraffin sections. It was proven that HBD are secreted in fairly large amounts in cases of chronic inflammation. Their secretion during the malignant transformation is limited. This is a very probable fact that plays a role in malignant transformation in tonsillar tissue. The crucial role in the development of chronic inflammation, and maybe that of malignant transformation, is played by eNOS and its product NO molecule. eNOS and the NO molecule are involved in cell cycle regulation, in the apoptotic processes and cell proliferation, as well as in the angiogenesis and vasculogenesis. Our result confirmed that eNOS is presented in the tissues of the upper airways in both chronic inflammation and carcinomatous processes. Ki-67 and cleaved caspase 3 were used as markers of cell proliferation and apoptosis.
- PublicationOpen AccessDifferential role of mesangial cells and podocytes in TGF-ß-induced mesangial matrix synthesis in chronic glomerular disease(Murcia : F. Hernández, 2009) Lee, Hyun Soon; Song, Chi YoungGlomerulosclerosis is characterized by mesangial matrix accumulation that is mediated primarily by activation of transforming growth factor-ß (TGF-ß). Unlike podocytes, mesangial cells secrete TGF-ß in response to common in vitro fibrogenic stimuli. However, mesangial immunostaining for active TGF-ß1 in chronic glomerular disease is almost negligible, despite increased mesangial TGF-ß1 mRNA expression, while podocytes covering the sclerotic glomerular segments exhibit increased TGF-ß1 protein expression. The mechanisms whereby TGF-ß is activated in the diseased glomeruli and how the activated TGF-ß leads to mesangial matrix overproduction are not clear. We provide evidence that TGF-ß secreted as latent complexes by mesangial cells is stored in the mesangial matrix, from which soluble forms of latent TGF-ß are released and localized to the podocyte surface in chronic glomerular disease. Podocyte-derived reactive oxygen species, plasmin and thrombospondin-1, particularly renin-angiotensin-aldosterone system-induced oxidative stress, seem to be involved in TGF-ß activation in podocytes. We also provide evidence that the TGF-ß- induced secretion of connective tissue growth factor and vascular endothelial growth factor by podocytes acts as a paracrine regulatory mechanism on mesangial cells, which may cause mesangial matrix accumulation culminating in the development of glomerulosclerosis. Collectively, these data bring new insights into our understanding of the roles of the mesangial cells and podocytes in the TGF-ß-induced mesangial matrix synthesis in chronic glomerular disease.
- PublicationOpen AccessPericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche(Murcia : F. Hernández, 2009) Díaz-Flores, Lucio; Gutiérrez, Ricardo; Varela, H.; Valladares, Francisco; Acosta, E.; Martín-Vasallo, P.; Díaz-Flores Jr., L.; Madrid Cuevas, Juan Francisco; Biología CelularWe review the morphofunctional characteristics of pericytes and report our observations. After a brief historical background, we consider the following aspects of pericytes: A) Origin in embryonic vasculogenesis (mesenchymal stem cells, neurocrest and other possible sources) and in embryonic and postnatal life angiogenesis (pre-existing pericytes, fibroblast/ myofibroblasts and circulating progenitor cells). B) Location in pericytic microvasculature and in the other blood vessels (including transitional cell forms and absence in lymphatic vessels), incidence (differences depending on species, topographical location, and type and stage of vessels) and distribution (specific polarities) in blood vessels. C) Morphology (cell body, and longitudinal and circumferential cytoplasmic processes), structure (nucleus, cytoplasmic organelles and distribution of microtubules, intermediate filaments and microfilaments) and surface (caveolae system). D) Basement membrane disposition, formation, components and functions. E) Contacts with endothelial cells (ECs) (peg and socket arrangements, adherent junctions and gap junctions) and with basal membrane (adhesion plaques). F) Molecular expression (pericyte marker identification). G) Functions, such as vessel stabilization, regulation of vascular tone and maintenance of local and tissue homeostasis (contractile capacity and vessel permeability regulation), matrix protein synthesis, macrophage-like properties, immunological defense, intervention in coagulation, participation in mechanisms that regulate the quiescent and angiogenic stages of blood vessels (including the behaviour of pericytes during sprouting angiogenesis and intussuceptive vascular growth, as well as pericyte interactions with endothelium and other cells, and with extracellular matrix) and plasticity, as progenitor cells with great mesenchymal potential, originating other pericytes, fibroblast/myofibroblasts, preadipocytes, chondroblasts, osteoblasts, odontoblasts, vascular smooth muscle and myointimal cells. This mesenchymal capacity is seen in a broad section on the perivascular mesenchymal cell niche hypothesis and in the concept of pericyte and EC “marriage and divorce”. H) Peculiar pericyte types, such as hepatic stellate cells (Ito cells), bone marrow reticular cells and mesangial cells. I) Involvement in pathological processes, such as repair through granulation tissue, pericyte-derived tumors, tumor angiogenesis and tumoral cell metastasis, diabetic microangiopathy, fibrosis, atherosclerosis and calcific vasculopathy, lymphedema distichiasis, chronic venous insufficiency, pulmonary hypertension, Alzheimer disease and multiple sclerosis. J) Clinical and therapeutic implications (de-stabilization of vessels or formation of a stable vasculature).
- PublicationOpen AccessUnexpected presence of the neurotrophins NGF and BDNF and the neurotrophin receptor p75 in the tendon cells of the human Achilles tendon(Murcia : F. Hernández, 2009) Bagge, Johan; Lorentzon, Ronny; Alfredson, Hàkan; Forsgren, StureNeurotrophins are substances that have been shown to be important in growth and remodelling phases in different types of tissue. There is no information concerning the possible occurrences of neurotrophins and their receptors in tendons. In this study, sections of both chronic painful (tendinosis) and pain-free (nontendinosis) human Achilles tendons were immunohistochemically stained with antibodies against the neurotrophins NGF and BDNF, and their receptors TrkA, TrkB and p75. There were marked immunoreactions for NGF and BDNF in the tendon cells (tenocytes) of both tendinosis and non-tendinosis specimens. The tenocytes were also reactive for the receptor p75, but not for the receptors TrkA and TrkB. In addition, p75 immunoreactions were seen in nerve fascicles and in the walls of arterioles. This is the first study to identify neurotrophins in the tenocytes of human tendon. It is clear from this study that the local cells of tendons are sources of neurotrophins. The neurotrophins may play an important role in the tendon through their interaction with the receptor p75 in the tenocytes. These interactions may regulate tropic modulatory, and apoptotic effects. In conclusion, the observations show a new concept concerning production and function of neurotrophins, namely in the tenocytes of tendons.
- PublicationOpen AccessOxidative stress, isoprostanes and hepatic fibrosis(Murcia : F. Hernández, 2009) Comporti, Mario; Arezzini, Beatrice; Signorini, Cinzia; Vecchio, Daniela; Gardi, ConcettaAn introduction to oxidative stress enlightening the spreading of interest in lipid peroxidation in the 60's and in the identification of cytotoxic aldehydes originating from it is given. The discovery of F2 -isoprostanes as specific markers of oxidative stress is described. Isoprostanes are also agonists of important biological effects. Since a relationship between oxidative stress and collagen hyperproduction has been previously suggested, and since lipid peroxidation products (aldehydes) have been proposed as possible mediators of liver fibrosis, we investigated whether collagen synthesis is induced by F2 -isoprostanes, which can posses receptors for signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma isoprostanes were markedly elevated for the entire experimental period and hepatic collagen content was also increased. Moreover, when hepatic stellate cells (HSC) isolated from normal livers were cultured up to activation and then treated with F2 -isoprostanes (8-epiPGF2α ) in the concentration range found in the in vivo studies (10-9 to 10-8 M), a striking increase in DNA synthesis, in cell proliferation and in collagen synthesis was observed. F2 -isoprostanes also increased the production of transforming growth factor-ß1 by U937 cells, assumed as a model of Kupffer cells or liver macrophages. The hypothesis that F2 -isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen hyperproduction, seen in this experimental hepatic fibrosis, was reinforced by the demonstration, by using immunoblot analysis, that isoprostane receptors identical or analogous to those for thromboxane A2 (TxA2 r) are present in HSC. Immunocytochemical studies showed the major localization of TxA2 r in the perinuclear site and its colocalization with α-smooth muscle actin.