Histology and histopathology Vol.28, nº 5 (2013)
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- PublicationOpen AccessQuantification of protein expression in cells and cellular subcompartments on immunohistochemical sections using a computer supported image analysis system(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Braun, Martin; Kirsten, Robert; Rupp, Niels J.; Moch, Holger; Fend, Falko; Wernert, Nicolas; Kristiansen, Glen; Perner, SvenQuantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual (‘eyeballing’) scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman’s rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p<0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.
- PublicationOpen AccessClusterin expression in elastofibroma dorsi(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Aigelsreiter, Ariane; Pichler, Martin; Pixner, Thomas; Janig, Elke; Schuller, Monika; Lackner, Carolin; Scheipl, Susanne; Beham, Alfred; Regauer, SigridBackground: Elastofibroma dorsi is a benign soft tissue lesion composed of abnormal elastic fibers. Degenerated elastic fibers in skin and liver are associated with clusterin, an apoprotein that shares functional properties with small heat shock proteins. We evaluated the staining pattern and possible role of clusterin in elastofibroma dorsi. Material and methods: Twenty-one subcutaneous elastofibromas from the scapular region were evaluated with Elastica van Gieson and Orcein stains, immunohistochemically with antibodies to clusterin, smooth muscle actin, S-100, vimentin and CD34 and correlated with clinical data with respect to physical trauma. Results: Clusterin correlated with the staining pattern of Elastica van Gieson and labelled abnormal broad coarse fibrillar and globular elastic fibers in all elastofibromas. Orcein stains additionally identified fine oxytalan fibers which were not stained by clusterin. Clusterin staining was observed only on the outside of the elastin fibers, while the cores of fibers and globules were unstained. 4/21 elastofibromas showed cellular nodules with a myxoid/ collagenous stroma. The round to oval cells showed cytoplasmic staining with vimentin and clusterin; CD34 labelled mostly cell membranes. The cells lacked SMA and S-100 expression. The central areas of the nodules were devoid of elastic fibers, but the periphery contained coarse fibers and globules. 9/11 patients, for whom clinical data were available, reported trauma to the scapular region. Conclusion: Many investigated ED were associated with trauma, which supports a reactive/ degenerative etiology of ED. The abnormal large elastic fibers in all ED were enveloped by clusterin. Clusterin deposition may protect elastic fibers from degradation and thus contribute indirectly to the tumor-like presentation of ED.
- PublicationOpen AccessMicroRNA expression profiles in metastatic and non-metastatic giant cell tumor of bone(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Mosakhani, Neda; Pazzaglia, Laura; Benassi, Maria Serena; Borze, Ioana; Quattrini, Irene; Picci, Piero; Knuutila, SakariGiant cell tumor of bone (GCTB) is a skeletal neoplasm, a locally aggressive tumor that occasionally metastasizes to the lungs. To identify novel biomarkers associated with GCTB progression and metastasis, we performed a miRNA microarray on ten primary tumors of GCTB, of which five developed lung metastases and the rest remained metastasis-free. Between metastatic and non-metastatic GCTB, 12 miRNAs were differentially expressed (such as miR-136, miR-513a-5p, miR-494, miR-224, and miR-542-5p). A decreased level of miR-136 in metastatic versus nonmetastatic GCTB was significantly confirmed by the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (p=0.04). To identify potential target genes for the differentially expressed miRNAs, we used three target prediction databases. Then, to functionally validate the potential target genes of the differentially expressed miRNAs, we re-analyzed our previous gene expression data from the same ten patients. Eight genes such as NFIB, TNC, and FLRT2 were inversely expressed relative to their predicted miRNA regulators. NFIB expression correlated in metastatic GCTB with no or low expression of miR-136, and this gene was selected for further verification with qRT-PCR and immunohistochemistry. Verification of NFIB mRNA and protein by qRT-PCR showed elevated expression levels in metastatic GCTBs. Further, the protein expression level of NFIB was tested in an independent validation cohort of 74 primary archival GCTB specimens. In the primary tumors that developed metastases compared to the disease-free group, NFIB protein was moderately to strongly expressed at a higher frequency. Thus, in GCTB, miR-136 and NFIB may serve as prognostic makers.
- PublicationOpen AccessIntrathyroid epithelial thymoma (ITET) and carcinoma showing thymus-like differentiation (CASTLE). CD5-positive neoplasms mimicking squamous cell carcinoma of the thyroid(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Kakudo, Kennichi; Bai, Yanhua; Ozaki, Takashi; Homma, Kei-ichi; Ito, Yasuhiro; Miyauchi, AkiraCarcinoma of possible thymic epithelial origin may occur within the thyroid gland, which was first reported by Miyauchi et al. as intrathyroid epithelial thymoma (ITET). ITET is a rare tumor comprising about 0.08% of all primary thyroid malignancies. It is a lowgrade thyroid carcinoma with squamous cell differentiation whose overall survival rate was found to be 71%. Lymph node metastasis at surgery was found in 40% and hematogenous metastases developed in bones, liver and lungs. This tumor grows within the thyroid gland and invades into the thyroid parenchyma as well as into the extrathyroid structures. It is a wellcircumscribed solid tumor with a sharp tumor border, but is not capsulated. After fixation, the cut surface of the tumor is gray-white in color and is a solid tumor with lobulation. Tumor calcification was not detected in our 15 cases. The tumor cells show solid sheets of growth with occasional keratinization without follicular or papillary structures. Lymphocytic infiltration in the stroma is one of the most characteristic features of this tumor. The tumor cells are polygonal epithelial cells with distinct nucleoli and ill-defined cell border. Positive immunoreactivity for CD5 is a key feature to differentiate it from undifferentiated carcinoma, poorly differentiated carcinoma, medullary (C cell) carcinoma and high-grade squamous cell carcinoma (so-called primary squamous cell carcinoma) of the thyroid. Negative immunoreactivity for calcitonin, TTF1 and thyroglobulin, and positive immunoreactivity for p63 and KIT are also helpful for differential diagnosis. Nuclear atypia is mild and mitoses are less frequent, with an intermediate proliferation index (MIB-1 labeling index is usually less than 20%), which are also helpful to differentiate it from high-grade primary squamous cell carcinoma of the thyroid. The tumors in our 15 cases demonstrate 3 histological subtypes: keratinizing squamous cell carcinoma type, non-keratinizing basaloid cell carcinoma (lymphoepithelioma-like) type and neuroendocrine carcinoma type, which correspond to subtypes of the mediastinal thymic carcinomas.
- PublicationOpen AccessImmunohistochemistry of cytokeratins 7, 8, 17, 18, and 19, and GLUT-1 aids differentiation of desmoplastic malignant mesothelioma from fibrous pleuritis(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Horiuchi, Toshikatsu; Ogata, Sho; Tominaga, Susumu; Hiroi, Sadayuki; Kawahara, Kunimitsu; Hebisawa, Akira; Irei, Isao; Ito, Ichiro; Kameya, Toru; Tsujimura, Tohru; Nakano, Takashi; Nakanish, Kuniaki; Kawai, ToshiakiIt is difficult to distinguish desmoplastic malignant mesothelioma (DMM) from fibrous pleuritis (FP). We investigated the utility of immunohistochemistry as a way of differentiating between DMM and FP. We examined 11 DMMs and 46 FPs with the aid of antibodies against 18 cytokeratin (CK) subtypes, calponin, caldesmon, desmin, and GLUT-1. The best sensitivity and specificity cut-off values in the receiver operating characteristic curves (ROC) for CKs 7, 8, 17, 18, and 19, and GLUT-1 were each above 60%. When cases with either DMM or FP were partitioned by the staining score associated with the best sensitivity and specificity cut-off values in ROC, the incidence of a positive expression for CKs 7, 8, 17, 18, and 19, and GLUT-1 was significantly higher in DMM than in FP. In conclusion, immunohistochemistry for CKs 7, 8, 17, 18, and 19, and GLUT-1 may be useful, alongside histological characteristics, for separating DMM from FP.
- PublicationOpen AccessThe foetal pig pineal gland is richly innervated by nerve fibres containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY, but it does not secrete melatonin(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Bulc, Michał; Lewczuk, Bogdan; Prusik, Magdalena; Całka, JaroslawInnervation of the mammalian pineal gland during prenatal development is poorly recognized. Therefore, immunofluorescence studies of the pineals of 70- and 90-day-old foetuses of the domestic pig were performed using antibodies against tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DßH), neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON). The investigated glands were supplied by numerous nerve fibres containing TH and DßH. The density of these fibres was higher in the distal and middle parts of the gland than in the proximal one. NPY and CPON were identified in the majority of DßH-positive fibres as well as in a small population of DßH-negative fibres localized mainly in the proximal part of the pineal. The immunoreactive fibres were more numerous in 90-dayold foetuses than in 70-day-old ones. The effect of norepinephrine on melatonin secretion by the foetal pineals in the short-term organ culture was studied to determine the role of DßH-positive fibres during prenatal life. For the same purpose melatonin was measured in the blood in the umbilical cords and in the jugular vein of the mother. The pineals of both groups of foetuses did not secrete melatonin in the organ culture, independently of the presence or absence of norepinephrine in the medium. Melatonin concentrations in the blood in the umbilical cords of foetuses from the same litter and in the jugular vein of their mother were similar. The presence of adrenergic nerve fibres in the pig pineal during gestation does not seem to be associated with the control of melatonin secretion.
- PublicationOpen AccessVEGF-B expression in colorectal carcinomas and its relevance for tumor progression(2013) Jayasinghe, Caren; Simiantonaki, Nektaria; Kirkpatrick, JamesThe biological behavior of VEGF-B, a ligand of the receptor VEGFR-1, is still enigmatic. Despite its high sequence homology to the better known angiogenetic factor VEGF-A, the function of VEGF-B has remained elusive. Especially, its role in tumor biology has thus far not been defined. In the present study we address the question of whether VEGF-B could play a role in the metastatic process of colorectal carcinomas (CRC). Using immunohistochemistry we investigated its expression in the tumor tissue of 91 nonmetastatic, lymphogenous-metastatic and haematogenous-metastastic CRC. Independently of metastatic status, VEGF-B was expressed in endothelial as well as in tumor cells. 81% of the CRC showed a positive, partly focal, partly disseminated endothelial expression in intratumoral vessels and the vascular fraction throughout the invasive tumor margin. Almost all of the VEGF-B positive vessels were of blood vascular origin. Many of these were thick-walled blood vessels with atherosclerotic changes characterizing preexistent but not angiogenetic vasculature. Thus it appears that VEGF-B might be an important ligand to ensure the blood supply for tumor survival. 46% of the CRC presented an additional tumoral VEGF-B expression which significantly correlated with haematogenous metastasis (p=0.006). These morphological results provide evidence for a probable pathobiological significance of VEGF-B in the tumor progression of CRC, especially in the case of haematogenous metastasis. It appears that VEGF-B might be an important ligand in the signalling between the tumor and preexisting blood vessels to ensure a functional blood supply for tumor survival.
- PublicationOpen AccessAdipose stem cells and skeletal repair(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Im, Gun-II. Although adipose tissue has been considered a useless tissue, recent investigations have shown that it provides an abundant source of adult stem cells. Adipose stem cells (ASCs) can undergo rapid osteogenenic differentiation, which represents a promising option for bone tissue engineering and treating large bone defects. While bone marrow-derived stem cells have been more extensively studied for bone tissue engineering, a limitation exists in the harvested amount of bone marrow. As adipose tissue can provide a much greater number of adult stem cells without causing morbidity, it offers a good option as a cell source for bone tissue engineering. In this review, we discuss the definition of ASCs, the induction of osteogenic differentiation from ASCs, scaffolding materials for adipose bone tissue engineering, and in vivo models for future clinical applications.
- PublicationOpen AccessEpigenetic alterations in colorectal cancer: the CpG island methylator phenotype(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Bae, Jeong Mo; Kim, Jung Ho; Kang, Gyeong HoonDNA methylation is one of the key mechanisms of epigenetic modification, and genomewide hypomethylation and CpG island hypermethylation are characteristics of cancer cells. The CpG island methylator phenotype (CIMP) is a distinctive subtype of colorectal cancers (CRCs) that show concordant hypermethylation of numerous promoter CpG island loci. CIMP-positive CRCs are associated with a proximal location in the colon, microsatellite instability, BRAF mutation and a relatively poor clinical outcome. CIMP-positive CRCs have their own precursor lesions, serrated adenomas, distinct from conventional adenomas which progress and transform into CIMP-negative CRCs. Although the existence of CIMP-positive CRCs is generally accepted, there has been controversy over technical issues with gene markers, the methodology used to define CIMP, and the prognostic or predictive role of CIMP. This review addresses recent advances in the field of CIMP-related research.
- PublicationOpen AccessThe level of phosphorylated Akt predominantly reflects the expressive status of CerbB2 in invasive breast cancer(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Chi, Yingkai; Zhao, Jing; Cui, Su-Ping; Jiang, Ping; Wang, Jian-ping; Zhang, Hong; Mao, Jing-Zhuo; Liu, Hai-Jing Liu; Hou, Lin; Zhang, BoAlthough some evidence has been documented on EGFR/PI3K mediation of Akt activation in breast cancers, ILK and DNA-PK have not been investigated so far. The aim of this study was to analyze the expression of phosphorylated Akt (pAkt) in breast cancer, with respect to its upstream regulators. The immunostaining of pAkt (Ser473) in 70 invasive breast cancers revealed that status of CerbB2 could play a major role in Akt phosphorylation, while ILK was also involved in the stimulated level of pAkt. The results would provide an important clue for the activation of Akt and potential targeted therapy in breast cancer.
- PublicationOpen AccessThe plantaris tendon in association with mid-portion Achilles tendinosis – Tendinosis-like morphological features and presence of a non-neuronal cholinergic system(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Spang, Christoph; Alfredson, Hàkan; Ferguson, Mark; Roos, Beverley; Bagge, JohanThe plantaris tendon is often neglected in morphological/clinical studies on the lower extremity. There is, however, clinical evidence that the plantaris tendon is involved in cases with Achilles midportion tendinopathy/tendinosis. It is nevertheless unclear if the plantaris tendon exhibits tendinosis-like features in this situation. We therefore investigated the plantaris tendon of patients with midportion Achilles tendinosis when the plantaris tendon was found to be located very close to or invaginated into the Achilles tendon, a situation which very often has been found to be the case. There was a very large number of tenocytes in the tendon tissue and the tenocytes showed abnormal and irregular appearances, exhibiting widened/rounded and wavy appearances, and were frequently lined up in rows. These features are characteristic features in Achilles tendinosis tendons. The tendon cells showed a distinct immunoreaction for the acetylcholine (ACh) -producing enzyme choline acetyltransferase (ChAT). Frequent fibroblasts were found in the loose connective tissue and these cells also showed a marked ChAT immunoreaction. The study shows that the plantaris tendon is morphologically affected in a similar way to the Achilles tendon in cases with midportion Achilles tendinosis and medial pain. The plantaris tendon may accordingly be a co-factor in these cases. The results also favour that there is a local ACh production both within the tendon tissue of the plantaris tendon and in the loose connective tissue. In conclusion, it is evident that plantaris tendons lying invaginated into or very close to the Achilles tendon in cases with midportion Achilles tendinosis show similar tendinosis features as previously shown for the Achilles tendon itself in these cases.
- PublicationOpen AccessIgG4-related disease: a systemic condition with characteristic microscopic features(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Detlefsen, SönkeDuring the first decade of the 21st century, IgG4-related disease (IgG4-RD), a fibroinflammatory condition occurring at multiple sites of the body, has been newly recognized. As indicated by its name, elevation of IgG4 in the serum and tissue is a common denominator of IgG4-RD. Since the observation that many patients suffering from autoimmune pancreatitis (AIP), a specific type of chronic pancreatitis, had elevated serum levels of IgG4, it was reported that these patients also had increased numbers of IgG4-positive cells in the inflamed pancreatic tissue. In 2003, it was noted that a significant proportion of the AIP patients had a variety of extrapancreatic fibroinflammatory lesions, and that AIP therefore was the pancreatic manifestation of a systemic disease. Among these extrapancreatic manifestations, the extrahepatic bile ducts, salivary glands, thyroid, lymph nodes and retroperitoneum were most frequently reported, and infiltration of the tissue with IgG4-positive cells was also noted at these sites. During the following years, a multitude of other conditions have been added to the spectrum of IgG4-RD. While some of these organ manifestations were once believed to represent diseases on their own, others have been included under the umbrella of "multifocal fibrosclerosis". Biopsies or resection specimens from affected organs in IgG4-RD reveal several common microscopic features irrespective of the site of the lesion. Cellular and storiform fibrosis, lymphoplasmacytic infiltration, increased numbers of IgG4-positive cells and obliterative phlebitis are among the most characteristic histological changes in IgG4-RD. The detailed etiology, pathophysiology, epidemiology and clinical long-term outcome have at present yet to be fully elucidated. This paper focuses on the microscopic features, diagnosis and differential diagnosis of the different organ manifestations of IgG4-RD, and the current concepts of its pathogenesis will also be addressed.
- PublicationOpen AccessShort-term behavior of different polymer structure lightweight meshes used to repair abdominal wall defects(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Pascual, G.; Hernández-Gascón, B.; Sotomayor, S.; Peña, E.; Calvo, B.; Buján, J.; Bellón, J.M.Background. While lightweight (LW) polypropylene (PP) meshes are been used for hernia repair, new prosthetic meshes also of low-density and with large pores have recently been introduced composed of other polymer materials. This study compares the behavior in the short-term of two macroporous LW prosthetic materials, PP and non-expanded PTFE. Methods. Partial defects were created in the lateral wall of the abdomen in New Zealand White rabbits and then repaired using a LW PP mesh or a new monofile, LW PTFE mesh. At 14 days postimplant, shrinkage and tissue incorporation, gene and protein expression of neocollagens (qRT-PCR/immunofluorescence), macrophage response (immunohistochemistry) and biomechanical strength were determined. Results. Both meshes induced good host tissue ingrowth, yet the macrophage response was significantly greater for the PTFE implants (p<0.05). Collagen 1/3 mRNA expression was greater for the PP mesh but differences lacked significance. Similar patterns of collagen I and III protein expression were observed in the neoformed tissue infiltrating the two meshes. After 14 days of implant, tensile strengths were also similar, while elastic modulus values were higher for the PTFE mesh (p<0.05). Conclusions. In the short term, host collagen deposition and biomechanical performance seemed unaffected by the polymer structure of the implanted mesh. In contrast, the inflammatory response to mesh implant produced at this early time point was more intense for the PTFE.