Histology and histopathology Vol.19, nº 1 (2004)

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Open Access
The ARF-p53 senescence pathway in mouse and human cells
(Murcia : F. Hernández, 2004) Wadhwa, R.; Sugahara, T.; Taira, K.; Kaul, S.C.
Mouse and human cells have most frequently been used for studies that have led to the elucidation of various molecular pathways involved in senescence. The ARF–p53 pathway has been assigned as one of the major protagonists in these phenomena. ARF is an alternative reading frame protein encoded along with p16INK4A by the INK4a locus on human chromosome 9p21 and the corresponding locus on mouse chromosome 4. Whereas the mouse ARF (p19ARF) consists of 169 amino acids, the human ARF (p14ARF) consists of 132 amino acids, truncated at the C-terminus. Molecular studies on the regulation of ARF activity by its binding partners have revealed that mouse ARF protein, but not human ARF protein, interacts with a cytoplasmic protein, Pex19p. This interaction of mouse ARF with Pex19p results in its milder p53 activation function in mouse cells as compared to human cells and thus accounts, at least in part, for the weaker tumor surveillance and frequent immortalization of mouse cells.
Open Access
Pathological change of articular cartilage in the mandibular head treated with immunosuppressant FK 506
(Murcia : F. Hernández, 2004) Ueno, T.; Kagawa, T.; Kanou, M.; Fujii, T.; Fukunaga, J.; Mizukawa, N.; Sugahara, T.; Yamamoto, T.
While several reports have documented immunosuppressant-induced osteoporosis, the exact mechanism of the pathological change of the joint remains to be clarified. In the present study, we have demonstrated the pathological change of the articular cartilage in the mandibular head of five Sprague-Dawley rats administered with the immunosuppressant FK 506 for 28 days. Three-dimensional micro-computed tomography of the mandibular heads in treated rats showed a significant decrease in trabecular bone volume compared to control rats. Histological observation revealed atrophic change of the articular cartilage. Immunohistological observation using anti-proliferative cell nuclear antibody (PCNA), type I, II, and type X collagen antibodies showed significantly decreased proliferation and differentiation of chondrocytes in the articular cartilage compared with the control group (p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant difference in the numbers of osteoclasts at the chondro-osseous junction. Thus, FK 506 administration inhibited chondrogenic cell proliferation and differentiation and might cause osteoporotic change of subcartilage trabecular bone that subsequently forms in the mandibular head
Open Access
Promoter methylation status of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia
(Murcia : F. Hernández, 2004) Tamura, G.
A number of tumor suppressor and tumorrelated genes exhibit promoter hypermethylation with resulting gene silencing in human cancers. In addition, several gene promoters have also been shown to become hypermethylated in non-neoplastic cells during aging. To assess the physiological consequence and clinical significance of gene promoter methylation in gastric epithelia, our laboratory has studied the methylation status of tumor suppressor and tumor-related genes, including APC, DAP-kinase, DCC, E-cadherin, GSTP1, hMLH1, p16, PTEN, RASSF1A, RUNX3 and TSLC1, in neoplastic and non-neoplastic gastric epithelia. The tumor suppressor and tumor-related genes, except APC, were generally unmethylated in non-neoplastic gastric epithelia obtained from younger individuals. The frequencies of methylation increased with age to varying degrees in various genes, although GSTP1 and PTEN methylation was completely absent in both neoplastic and non-neoplastic gastric epithelia. The methylation frequencies in each gene were found to be comparable in neoplastic and non-neoplastic gastric epithelia, except the methylation of RUNX3 and TSLC1, which was mostly cancer-specific (P<0.01). When methylation frequencies were compared between non-neoplastic gastric epithelia from cancer-bearing and non-cancerbearing stomachs, hMLH1 and p16 methylation was more frequent in those from cancer-bearing stomachs (P<0.01). Promoter methylation in tumor suppressor and tumor-related genes initially occurs in non-neoplastic gastric epithelia, increases with age, and ultimately silences gene function to constitute a field-defect that may predispose tissues to gastric cancer evolution. In clinical applications RUNX3 and TSLC1 methylation may be utilized as molecular diagnostic markers, and hMLH1 and p16 methylation as predictors of malignancy in the stomach.
Open Access
The distribution and role of myofibroblasts and CD34-positive stromal cells in normal pancreas and various pancreatic lesions
(Murcia : F. Hernández, 2004) Kuroda, Naoto; Toi, M.; Nakayama, Hiroyuki; Miyazaki, E.; Yamamoto, M.; Hayashi, Yoshihiro; Hiroi, Makoto; Enzan, H.
To elucidate the distribution and role of myofibroblasts and CD34-positive stromal cells in various pancreatic lesions, we performed an immunohistochemical study using a streptoavidin-biotin immunoperoxidase technique. We selected 43 pancreatic lesions from 1 biopsied, 22 surgically resected and 12 autopsied specimens: acute pancreatitis (n=3), chronic non-obstructive pancreatitis (n=4), obstructive pancreatitis (n=7), islet cell tumor (n=4), serous cystadenoma (n=7), mucinous cystadenoma (n=6), and invasive ductal carcinoma (n=12). In normal pancreas, myofibroblasts and CD34-positive stromal cells were predominantly present in the peridcutal and periacinar areas, respectively. Both myofibroblasts and CD34- positive cells were observed in the stroma of chronic pancreatitis. In four islet cell tumors, myofibroblasts were present in the stroma of the tumor center, but no CD34-positive stromal cells were identified. Additionally, myofibroblasts and CD34-positive stromal cells were located in the inner layer and the outer layer of the capsule of three islet cell tumors, respectively. In nine of the thirteen cystadenomas, only myofibroblasts were recognized in the cyst wall. In the remaining four cystadenomas, a small number of CD34-positive cells were observed in the cyst wall. In 12 invasive ductal carcinomas, the stroma possessed a lot of myofibroblasts, but there were no or few CD34-positive stromal cells. In conclusion, it seems that the abundant amount of CD34-stromal cells in the main lesions is characteristic of chronic inflammatory lesions. Myofibroblasts and CD34-positive stromal cells may play a role in regulating the tumor growth in the capsule of islet cell tumors of the pancreas.
Open Access
Skeletal muscle regeneration and Trypanosoma cruzi-induced myositis in rats
(Murcia : F. Hernández, 2004) Maldonado, I.R.S.C.; Ferreira, M.L.; Camargos, E.R.S.; Chiari, E.; Machado, C.R.S.
Although Chagas’ disease is known to provoke severe acute myositis, information on muscle regeneration is missing. The current paper shows that during T. cruzi infection in rats, skeletal muscle parasitism and the consequent inflammatory process are higher in muscle with a high proportion of type-I myofibres (soleus and diaphragm). Immunohistochemistry showed an acute inflammatory process characterized by ED1+ and ED2+ macrophages, CD8+ lymphocytes, and NK cells. Parasite-nest rupture provoked segmental degeneration of myofibres followed by regeneration. These phenomena were observed at both light and transmission electron microscopy levels. Myofibre regeneration involved activation of satellite cells assessed by the expression of MyoD, a musclespecific transcription factor. Ultrastructural evidence of fusion of myoblast-like cells with the intact segment of degenerating fibres has been provided. At the chronic phase no signs of fibrosis were found, but sparse and small inflammatory foci were found. Our results argue against the relevant participation of autoimmunity phenomena in both acute and chronic phases and furnish a new view for explaining histopathological findings in human patient muscles.
Open Access
Therapy-related changes of the bone marrow in chronic idiopathic myelofibrosis
(Murcia : F. Hernández, 2004) Thiele, J.; Kvasnicka, H.M.; Schmitt-Gräff, A.; Hülsemann, R.; Diehl, V.
In chronic myeloproliferative disorders (CMPDs) a conflict of opinion exists regarding therapyinduced bone marrow (BM) changes and the evolution of myelofibrosis during the lengthy course of the disease. For a more elaborate study of these features chronic idiopathic myelofibrosis (IMF) seems to be a most suitable condition. Therefore this review is focused on this CMPD and amongst other findings analyzes data from a series of 340 patients with a long follow-up including 893 biopsies (median interval of 32 months). The ensuing results were compared with those communicated in the relevant literature. In addition to a control group of 153 patients with IMF who received only symptomatic treatment, therapy groups included busulfan, hydroxyurea, interferon and various combinations. In all groups hypoplasia of a varying degree was a frequent finding (6%) and often accompanied by a patchy arrangement of hematopoiesis. Most conspicuous was a gelatinous edema showing a tendency to develop a discrete reticulin fibrosis (scleredema). Aplasia developed in 7.7% of patients, usually at terminal stages of the disease independently of treatment. Minimal to moderate maturation defects of hematopoiesis involved especially megakaryocytes and erythroid precursors, but overt myelodysplastic features were most prominent following hydroxyurea and busulfan therapy. Acceleration and blastic crisis were characterized not only by increasing dysplastic changes, but also by the appearence of blasts including CD34+ cells. Semiquantitative grading of the fiber content revealed that 183 patients (54%) without or with moderate fibrosis at the beginning showed a significant progression and therefore contrasted with the 66 patients with a stable state. Following this calculation no relevant differences in the evolution of myelofibrosis were evident in the various therapy groups especially not following interferon treatment. In a few patients a regression was found which was accompanied by a severe hypoplasia or aplasia compatible with a myeloablative effect. In conclusion, peculiar BM changes, in particular conspicuously expressed myelodysplastic features are consistent with therapy-related lesions. Development of myelofibrosis in IMF is obviously due to disease progression unrelated to stage at diagnosis and not significantly influenced by treatment modalities.
Open Access
The prognostic significance of thymidine phosphorylase, thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expressions in breast carcinomas
(Murcia : F. Hernández, 2004) Li, H.; Suo, Z.; Zhang, Y.; Risberg, B.; Karlsson, M.G.; Villman, K.; Nesland, Jahn M.
Thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have been indicated as possible predictive markers for epithelial malignancies. All these three enzymes are actively involved in 5-FU metabolism. In this report, we investigated mRNA expression of these factors with real-time quantitative PCR in a series of 86 micro-selected breast carcinomas and 8 micro-selected tumour-adjacent normal breast epithelial specimens. Highly variable mRNA expressions of these factors were observed in both normal and cancerous samples. TP and TS mRNA expressions in breast carcinomas were elevated, but only TS mRNA expression showed a trend for statistical difference, compared with the expression in normal breast epithelial samples. Although the DPD mRNA expression range in tumours was also elevated, the average mean was reduced in tumours compared to that in normal samples. No association between mRNA expressions of TP, TS and DPD and clinicopathological features such as histological grade, tumour size, node status, S-phase fraction, ploidy, and clinical stage was found. A negative association between DPD mRNA expression and age was, however, revealed. Ten-year follow-up analysis showed no association between TP and DPD mRNA expression and clinical outcome. An high level of TS mRNA expression, however, was associated with a shorter clinical survival, indicating its potential role as a clinical marker in breast carcinoma.
Open Access
Expression of vascular endothelial growth factor (VEGF) and association with microvessel density in small-cell and non-small-cell lung carcinomas
(Murcia : F. Hernández, 2004) Stefanou, D.; Batistatou, Anna; Arkoumani, E.; Ntzani, E.; Agnantis, N.J.
Recent studies have demonstrated that tumor angiogenesis is a prognostic factor for various malignant neoplasms. Specifically, in non-small-cell lung carcinomas (NSCLCs) most reports show an association between neovascularization and vascular endothelial growth factor (VEGF) expression as well as the presence of metastases and survival, although a few reports do not agree with these findings. Angiogenesis is not clearly characterized in small-cell lung carcinomas (SCLCs), since they are rarely treated by surgery, and thus the available tissue for biological characterization is sparse. The aim of the present study was to investigate angiogenesis and the expression of VEGF in lung tumors. We examined 88 non-small-cell and 39 smallcell lung carcinomas. Angiogenesis was estimated by determining microvessel counts, with the use of anti- CD31 and anti-factor VIII antibodies and expression of VEGF was also evaluated immunohistochemically. Our data showed that in NSCLCs angiogenesis was more prominent in poorly-differentiated neoplasms and correlated with VEGF expression, therefore it is at least in part mediated by the latter. Interestingly, in SCLCs a higher vascularization was noted. However, there was no strong association with VEGF expression. Thus, smallcell lung carcinoma may represent a suitable neoplasm for testing antiangiogenic drugs in combination with chemotherapy. Nevertheless, antiangiogenic therapy should not be targeted specifically to the VEGF pathway, since in SCLCs other mediators of angiogenesis may be important as well.
Open Access
Histopathology, pathogenesis and molecular genetics in primary central nervous system lymphomas
(Murcia : F. Hernández, 2004) Nakamura, M.; Shimada, K.; Ishida, E.; Konishi, N.
Recent increases in the incidence of primary central nervous system lymphoma (PCNSL), a rare non- Hodgkin’s lymphoma arising in the brain, have been noted in both immunodeficient and immunocompetent patients. Compared with lymphomas originating outside the central nervous system, the biology of PCNSL at the molecular or cytogenetic level has not been well characterized, yet it is important to thoroughly understand the etiology of this rare malignant lymphoma if effective therapies are to be developed. This review will focus on the epidemiology, clinical aspects, histopathology, pathogenesis, and molecular genetics of this aggressive, extranodal lymphoma in immunocompetent patients.
Open Access
Post-genomic applications of tissue microarrays: basic research, prognostic oncology, clinical genomics and drug discovery
(Murcia : F. Hernández, 2004) Mobasheri, A.; Airley, R.; Foster, C.S.; Schulze-Tanzil, G.; Shakibaei, M.
Tissue microarrays (TMAs) are an ordered array of tissue cores on a glass slide. They permit immunohistochemical analysis of numerous tissue sections under identical experimental conditions. The arrays can contain samples of every organ in the human body, or a wide variety of common tumors and obscure clinical cases alongside normal controls. The arrays can also contain pellets of cultured tumor cell lines. These arrays may be used like any histological section for immunohistochemistry and in situ hybridization to detect protein and gene expression. This new technology will allow investigators to analyze numerous biomarkers over essentially identical samples, develop novel prognostic markers and validate potential drug targets. The ability to combine TMA technology with DNA microarrays and proteomics makes it a very attractive tool for analysis of gene expression in clinically stratified tumor specimens and relate expression of each particular protein with clinical outcome. Public domain software allows researchers to examine digital images of individual histological specimens from TMAs, evaluate and score them and store the quantitative data in a relational database. TMA technology may be specifically applied to the profiling of proteins of interest in other pathophysiological conditions such as congestive heart failure, renal disease, hypertension, diabetes, cystic fibrosis and neurodegenerative disorders. This review is intended to summarize the strengths and weaknesses of TMA technology which will have an increasingly important role in the laboratories of the post-genomic era.
Open Access
Plasticity and regulation of human bone marrow stromal osteoprogenitor cells: potential implication in the treatment of age-related bone loss
(Murcia : F. Hernández, 2004) Ahdjoudj, S.; Fromigué, O.; Marie, P.J.
Human bone marrow stroma contains pluripotent mesenchymal progenitor cells that can give rise to many mesenchymal lineages, including chondroblasts, adipocytes or osteoblasts. The differentiation of these cells towards a specific lineage is dependent on hormonal and local factors activating specific transcription factors. Attempts have been recently made to identify osteoprogenitor cells in the human bone marrow and to identify the molecular mechanisms responsible for lineage-specific differentiation of human bone marrow stromal cells. Using a clonal pluripotent human bone marrow stromal cell line with tri-potential characteristics, we have provided evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of human bone marrow stromal cells. We have also shown that administration of TGFß that regulates the expression of specific osteoblast and adipocyte transcription factors can promote osteoblast differentiation and inhibit adipocyte conversion of rat marrow stromal cells in vivo. This indicates that the reciprocal relationship between osteoblastogenesis and adipogenesis can be manipulated in vivo in order to improve bone formation. Future studies will have to identify key signals for lineage-specific differentiation of human marrow stromal cells. This may result in the development of therapeutic strategies to promote the differentiation of these cells towards the osteoblast lineage and to inhibit excessive bone marrow adipogenesis associated with aging.
Open Access
The retinal pigment epithelium of the teleost Notopterus notopterus (Pallas): Appearance of basal infoldings during prolonged dark-adaptation
(Murcia : F. Hernández, 2004) Nag, T.C.
In teleosts, the basal part of the retinal pigment epithelium (RPE) is relatively smooth, i. e., it is free of basal membrane infoldings. In the featherback, Notopterus notopterus, whereas this is the situation in light adaptation, during dark-adaptation, especially when kept for prolonged periods (6-9 hour), numerous infoldings appear at the basal region, as found uniquely by transmission electron microscopy. In this teleost, during retinomotor movements, the rods move vitreally during dark-adaptation, while the cones do not elongate, and remain stationary in both light- and dark- adaptation. The significance of the appearance of basal infoldings in dark-adapted RPE is explained in terms of the pattern of retinomotor responses and the features of RPE and photoreceptors in this species. It is suggested that (1) the thick, impervious tapetal layer present in the RPE, (2) the unusual position of the photoreceptors in the visual cell layer of dark-adapted retina, and (3) the presumably high demand for glucose and O2 of the outer retina during dark-adaptation might contribute to cause this phenomenon in this species. The available evidence tend to associate this phenomenon with the involvement of the RPE in nutrient and O2 delivery to the photoreceptors via the basal infoldings of the RPE in dark-adapted state in this species. This has not been reported for any other teleosts to date.
Open Access
Recent advances in osteoclast biology and pathological bone resorption
(Murcia : F. Hernández, 2004) Blair, H.C.; Athanasou, N.A.
The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFa, interleukin-1, TGFß and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFa, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKLindependent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+- ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.
Open Access
Structural patterns of swine ileal mucosa following L-glutamine and nucleotide administration during the weaning period. An histochemical and histometrical study
(Murcia : F. Hernández, 2004) Domeneghini, C.; Di Giancamillo, A.; Savoini, G.; Paratte, R.; Bontempo, V.; Dell’Orto, V.
Dietary supplementations with L-glutamine and/or nucleotides were screened for their effects on intestinal mucosa in 16 female weaning piglets. The animals were transported to the university’s facilities 24 hours after weaning. They were grouped four to a pen in controlled environmental conditions and fed one of the following four diets for 28 days: control diet (C); C+0.5% L-glutamine (G); C+0.05% “nucleotides” (N); and C+0.5 % L-glutamine+0.05% “nucleotides” (GN). Individual body weights and feed intake per group were recorded at the beginning and the end of the study as well as weekly during it. There were no significant performance differences among the groups. After 28 days the animals were slaughtered and the distal ileum and liver were examined histologically. Antiproliferating cell nuclear antigen (PCNA) as well as antihuman macrophage immunostaining, and a modified TdT-mediated dUTP nick-end labeling technique (TUNEL) were performed, and intraepithelial lymphocyte percentage was evaluated to assess morphofunctional aspects of the ileum. Histometry was performed by assessing cell indices and counts of immuno-reactive structures. Feeding G and/or N resulted in an increase in villi (V) height, crypt (C) depth, and a decrease in V:C ratio P<0.01). In addition, feeding G and/or N resulted in an increase in mitotic mucosal cells (M), and a decrease in apoptotic mucosal cells (A), thus decreasing the A:M index (P<0.01). The percentages of mucosal macrophages were greater in G and/or N groups (P<.001) than in control piglets, and similarly among the groups the percentages of intraepithelial lymphocytes varied (P<0.01). Our data showed that the diet supplementation with G and/or N had positive effects on some morphofunctional characteristics of piglet ileal mucosa. These ameliorative effects may potentially be linked to a good responsiveness of piglets to a stressful period, like a precocious weaning is in this species.
Open Access
Expression and significance of cell immunohistochemical markers (HHF-35, CD-31, Bcl-2, P-53 and apopDETEC®) in hypertrophic cardiomyopathy
(Murcia : F. Hernández, 2004) Martínez Díaz, F.; Bernal-Gilar, M.; Gómez Zapata., Maximiliano; Luna Maldonado, Aurelio
There are several hypotheses concerning the pathogenesis of hypertrophic cardiomyopathy (genetic, ischaemic, immune, inflammatory and apoptosis induction). We have studied three types of cardiomyopathy in order to observe the expression and assess the significance of different immunohistochemical markers (muscular actin, CD-31, proliferation cell nuclear antigen -PCNA-, Ki-67, and markers related with programmed cell death, bcl-2, p-53 and apopDETEC®). We studied different microscopic (haematoxylineosin and Masson’s thrichrome) and immunohistochemical parameters (streptavidin-biotin-peroxidase and “in situ” hybridisation) of forty cases: ten each of hypertensive hypertrophic cardiomyopathy, essential hypertrophic cardiomyopathy, hypertrophic cardiomyopathy in patients treated with chemotherapy and morphologically “normal” hearts. Our findings point to an absence of structural marker expression (actin and CD-31) in cases of hypoxic damage. The distribution and intensity of apoptosis markers, a seen by “in situ” hybridisation were irregular, and the rest of the markers studied showed negative results, with the exception of acridin orange (a marker of hypoxic damage). In our opinion, the above immunohistochemical markers, especially actin and CD-31, could be used for differentiating hypoxic lesions in these three types of cardiomyopathy. Moreover, it is difficult to know the significance of the apoptosis markers, because the autolysis process produces cross reactions with false positive results. We think that there is a need for new studies on DNA breakdown processes during the postmortem interval. To avoid autolysis problems the postmortem material needs to be as fresh as possible.
Open Access
Apoptosis of thymocytes in experimental African Swine Fever virus infection
(Murcia : F. Hernández, 2004) Salguero, F.J.; Sánchez-Cordón, P.J.; Sierra, M. A.; Jover, A.; Núñez, A.; Gómez-Villamandos, J. C.
This paper report on the lesions occurred in the thymus in experimental acute African swine fever (ASF). Twenty-one pigs were inoculated with the highly virulent ASF virus (ASFV) isolate Spain-70. Animals were slaughtered from 1 to 7 days post infection (dpi). Three animals with similar features were used as controls. Thymus samples were fixed in 10% buffered formalin solution for histological and immunohistochemical study and in 2.5% glutaraldehyde for ultrastructural examination. For immunohistochemical study, the avidin-biotin-peroxidase complex (ABC) technique was used to demonstrate viral protein 73 and porcine myeloid-histiocyte antigen SWC3 using specific monoclonal antibodies. Cell apoptosis was evaluated by the TUNEL assay. Blood samples were taken daily from all pigs and were used for leukocyte counts. The results of this study show a severe thymocyte apoptosis not related to the direct action of ASFV on these cells, but probably to a quantitative increase in macrophages in the thymus and their activation. A decrease in the percentage of blood lymphocytes was observed at the same time No significant vascular changes were observed in the study. With these results we suggest that ASFV infection of the thymus does not seem to play a critical role in the acute disease. Although severe apoptosis was observed, animals died because of the severe lesions found in the other organs.
Open Access
Multicolor FISH probe sets and their applications
(Murcia : F. Hernández, 2004) Liehr, T.; Starke, H.; Weise, A.; Lehrer, H.; Claussen, U.
Multicolor fluorescence in situ hybridization (FISH) assays are nowadays indispensable for a precise description of complex chromosomal rearrangements. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Since then different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been described. Predominantly, they have been established to characterize marker chromosomes identified in conventional banding analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at the identification of disease-related genes. Here we present a review on the available mFISH methods including their advantages, limitations and possible applications.
Open Access
Immunohistochemical identification of intracytoplasmic lumens by cytokeratin typing may differentiate renal oncocytomas from chromophobe renal cell carcinomas
(Murcia : F. Hernández, 2004) Kuroda, Naoto; Toi, M.; Yamamoto, M.; Miyazaki, E.; Hayashi, Yoshihiro; Hiroi, Makoto; Shuin, T.; Enzan, H.
Renal oncocytomas and chromophobe renal cell carcinomas (RCCs) share a common phenotype and both originate from the intercalated cells of the collecting duct. This makes it very difficult to differentiate between the two tumors immunohistochemically. Therefore, we studied the results of immunohistochemistry focusing on certain characteristic structures that are occasionally present in renal oncocytomas. We carried out Hale’s colloidal iron staining and immunohistochemistry for various cytokeratins (cytokeratins 7, 8, 10, 10/13, 14, 18, 19 and 20, and AE1/AE3) in four oncocytomas and six chromophobe RCCs. In addition, one renal oncocytoma and one chromophobe RCC were studied using electron microscopy. Two renal oncocytomas and one chromophobe RCC were completely unstained by colloidal iron. There was no evident difference between the immunohistochemical characteristics of oncocytomas and those of chromophobe RCCs. However, in all four renal oncocytomas we identified intracytoplasmic ring-like positive reactions for some cytokeratins (at least 3 antigens of cytokeratins 7, 8 and 19, and AE1/AE3), which corresponded ultrastructurally to the intracytoplasmic lumens (ICLs). In contrast, no such structures were found in any of the chromophobe RCCs using the antibodies employed. Therefore, immunohistochemical identification of ICLs by cytokeratin typing may be useful for differentiating between renal oncocytomas and chromophobe RCCs and be more sensitive in this respect than colloidal iron staining.
Open Access
The effects of low laser irradiation on angiogenesis in injured rat tibiae
(Murcia : F. Hernández, 2004) Garavello, I.; Baranauskas, V.; da Cruz Hoflingl, M.A.
The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm-2 were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm-2, but significantly decreased the number of vessels in the 14- day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.
Open Access
Immunohistochemical study of the upper surface layer in rat mandibular condylar cartilage
(Murcia : F. Hernández, 2004) Zea-Aragón, Z.; Ohtsuki, K.; Ohnishi, M.; Ohno, S.
Both hyaluronic acid and fibronectin localizations were examined in the upper surface layer of rat mandibular condylar cartilages by immunohistochemical techniques. Their delicate structure was successfully preserved by preparation procedures of joint condyles with disks. Paraformaldehyde-fixed cartilaginous tissues were cut in a cryostat, and cryosections were analyzed using streptavidinperoxidase and indirect immunofluorescence methods. Another immunogold method with conventional preparation procedures and a quick-freezing method was performed for their ultrastructural analyses. Both hyaluronic acid-binding protein and anti-fibronectin antibody were used to localize hyaluronic acid and fibronectin in the mandibular condylar cartilage, respectively. Some cryosections were pre-treated with hyaluronidase and chondroitinase before such labeling. The upper surface layer was composed of double laminar structures. One bordered with the cartilage matriceal surface, which was positive for fibronectin. The hyaluronic acid was localized over the fibronectin layer. Therefore, the hyaluronic acid in vivo was bound with fibronectin in the cartilaginous matrix, performing lubrication for the mandibular joint movement.