Histology and histopathology Vol.22, nº 4 (2007)
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- PublicationOpen AccessAnti-l-light chain-peptide antibodies are suitable for the immunohistochemical classification of AL amyloid(Murcia : F. Hernández, 2007) Kuçi, H.; Ebert, M.; Röcken, C.We aimed to test whether antibodies raised against recombinant peptides corresponding to the variable region of immunoglobulin light chains are suitable for the immunohistochemical classification of amyloid. The Entrez database of the National Center for Biotechnology Information (NCBI) was searched for all protein sequence entries which met the search criteria “amyloid” and “lambda light chain”. Sixty-four different l-light chain-derived amyloid protein sequences were retrieved, aligned and categorized into the V region subgroups of l-light chain detailed by the NCBI, i.e. subgroup I (21 protein sequences), II (14), III (6), IV (1), V (1) and VI (21). V region subgroup I was chosen for epitope sequence selection and two rabbits were immunized with the following peptides: NH2- ISCSGSSSNIGSNTV-CONH2 and NH2-QRPSG VPDRFSGSKSGTS-CONH2. Sensitivity and specificity of the IgG-purified antibodies was tested by Western blotting using amyloid A- (AA), ALl- and ALk-amyloid proteins, and by immunohistochemistry on tissue microarrays with 110 different amyloid containing tissue samples obtained at autopsy from 22 patients, and on 27 biopsy specimens from a series of 24 patients. Our peptide antibodies specifically stained AL amyloid l- light chain-origin, in both Western blots and formalinthat peptide-antibodies directed against immunoglobulin- derived l-light chain proteins can be applied for the immunohistochemical classification of amyloid. This offers the opportunity to generate a large set of anti- l-light chain protein-antibodies for the immunohistochemical classification of amyloid independently from native human tissue sources.
- PublicationOpen AccessMicroencephaly and microphthalmia in rat fetuses by busulfan(Murcia : F. Hernández, 2007) Furukawa, S.; Usuda, K.; Abe, M.; Ogawa, I.Microencephaly and microphthalmia in the embryos/fetuses from rats exposed to busulfan were histopathologically examined. Busulfan was intraperitoneally administered at 10 mg/kg on gestation days (Days) 12, 13 and 14, and then embryos/fetuses were harvested on Days 14.5, 15, 16 and 21. In the treated group on Day 21, all fetuses were small with reduced body weight, with microencephaly and microphthalmia. On Days 14.5, 15 and 16, apoptotic cells were increased in the neuroepithelium and the neural retina with a width reduction and a decrease in cell density, and the lens epithelial cells histopathologically. Mitotic inhibition was observed in the neuroepithelium, neural retina and equatorial zone of the lens. On Day 21, the cerebral cortex and the retina became markedly thinner. The lens fibers showed swollen, fragmentary and vacuolar formation in the cranial portion accompanied with small lens sizes. The anti-proliferative effects of busulfan brings about a lack of cell populations required for the normal organogenesis of the brain and eye, and leads to microencephaly and microphthalmia, featuring hypoplasia of cerebrum and hypoplasia of retina and lens with cataract, respectively
- PublicationOpen AccessGel-forming mucins. Notions from in vitro studies(Murcia : F. Hernández, 2007) Perez-Vilar, J.; Mabolo, R.Mucus secretions form a protective barrier in the mucosa of the auditory, gastrointestinal, respiratory, and urogenital systems, and the conjunctiva in the eyes. A family of glycoproteins known as gel forming mucins is the major component of the mucus. Gel-forming mucins are among the largest and most complex proteins known. Their polypeptide chains comprise thousands of amino acid residues organized into different domains with diverse post-translational modifications, including O- and N-glycosylation, sulfation, proteolysis, and likely C-mannosylation. Moreover, these glycoproteins form disulfide-linked oligomers/multimers with molecular weights in the millions. Molecular polydispersity in terms of length, carbohydrate content and composition, is an invariable feature of purified mucins. This structural complexity makes it technically very difficult to study mucin biochemical and physical properties. It is not surprising, therefore, that our knowledge on mucin structure, biosynthesis and function still is incomplete. During the last decade, the use of recombinant mucins has allowed researchers to study the biochemical properties of protein domains, peptide motifs and amino acid residues common to all gel-forming mucins, and to propose specific roles for them. We review here the relative impact that these in vitro studies have had for our current understanding of two of the most important features of these macromolecules: formation of disulfide linked oligomers and mucin intragranular packaging.
- PublicationOpen AccessPrimary mammary osteogenic sarcoma(Murcia : F. Hernández, 2007) Khaldi, L.; Athanasiou, E.T.; Hadjitheofilou, C.Th.A 78 year-old female patient underwent a total mastectomy with axillary lymph node dissection for a primary breast osteosarcoma. Microscopically the tumor was identical to grade II skeletal osteosarcoma. Immunohistochemically no reactivity was detected, either for the epithelial markers EMA, AE1/AE3, CK8, 18, 19, or for HER-2/neu, estrogen and progesterone receptors, as well as fluorescent IN SITU hybridization for HER-2/neu. The diagnosis of this tumor fulfills certain clinicopathological criteria. Mammary osteosarcoma is usually developed in phyllodes tumors or carcinosarcomas of the breast as a result of metaplasia of the epithelial component. This rare tumor of the breast is occasionally associated with prior radiation therapy or well documented trauma. Mammary osteosarcoma is a biologically aggressive neoplasm with a 38% five-year survival rate. Surgical resection is the most effective therapy to date. Adjuvant treatment -chemotherapy or radiotherapy- has shown no clear benefit. An extensive review of the literature is also presented.
- PublicationOpen AccessShort term regulation of hepatocyte glutathione content by hepatic sinusoidal cells in co-culture(Murcia : F. Hernández, 2007) Catalá-Rodríguez, M.; Pagani, R.; Portolés, M.T.antioxidant mechanisms may constitute the primary mechanisms of a number of pathologies. The liver plays a central role in this balance: parenchymal hepatic cells contain and export especially high levels of the antioxidant glutathione and activated Kupffer cells release inflammation mediators and reactive oxygen species. There is growing evidence of a paracrine regulation of hepatic function by means of a fluent intercellular communication which must still be fully elucidated, especially in basal conditions. In vivo models provide often too complex results but, in vitro, tissue interactions are left aside; therefore it is important to find new experimental models to address cell communication studies. Here we propose the complementary use of three models to study liver glutathione system regulation in basal conditions: pure parenchymal cells primary cultures, addition of sinusoidal cell conditioned media to parenchymal cells and co-culture of sinusoidal cells using porous membranes. We have also developed a high specifity immunofluorescent method for the complete characterization of sinusoidal cell populations by flow cytometry and confocal microscopy. Our results show that Kupffer cells possess higher levels of reactive oxygen species than sinusoidal endothelial cells even in basal conditions. We also report that the glutathione content of hepatic parenchymal cells in basal conditions and suggest the existence of a paracrine circuit in the management of liver oxidative stress.
- PublicationOpen AccessNonsteroidal anti-inflammatory drugs and oxidative stress in cancer cells(Murcia : F. Hernández, 2007) Adachi, M.; Sakamoto, H.; Kawamura, R.; Wang, W.; Imai, K.; Shinomura, Y.Nonsteroidal antiinflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast and leukemia. In addition, the classical NSAIDs sulindac and aspirin are promising chemopreventive agents against colon cancer. NSAIDs inhibit cyclooxygenases (COX) preventing the formation of prostaglandins, prostacyclin and thromboxane. NSAIDs also exert other biological effects, including generation of reactive oxygen species (ROS) and inhibition of NF-kB-mediated signals. Despite many suggested mechanisms for their anticancer effects, it remains uncertain how they induce cell cycle arrest and apoptosis in cancer cells. Furthermore, there is little information on the selectivity of NSAIDs-mediated anticancer effects, although this is one of the most important issues in cancer therapy. Increased understanding of the biological basis for the anticancer activity of NSAIDs and their selectivity is essential for future therapeutic advances. In this paper, we propose that increased ROS generation is one of the key mechanisms for NSAIDs-mediated anticancer effects on various cancer cells.
- PublicationOpen AccessProtein networking in bladder cancer: Immunoreactivity for FGFR3, EGFR, ERBB2, KAI1, PTEN, and RAS in normal and malignant urothelium(Murcia : F. Hernández, 2007) Røtterud, R.; Fosså, S.D.; Nesland, Jahn M.A panel of markers, selected for the suspected bladder cancer relevance of their corresponding genes, were explored for their expression and subcellular location in urinary bladder tissue. The expression in normal urothelium, in non-metastasised transitional cell carcinomas (TCC), and in primary metastasised TCC with corresponding metastases was mapped. Potential associations between the proteins were identified. The observations were then combined in a set of hypotheses aimed at further hypothesis testing. Membranous ERBB4 and cytoplasmic p21RAS were downregulated in carcinoma cells compared with normal urothelium cells. FGFR3 was translocated from the cytoplasm to the nucleus. ERBB2 was translocated to the membrane and seemingly upregulated in one subgroup and conversely downregulated in another. EGFR, KAI1 and possibly PTEN revealed increased membranous immunoreactivity in non-metastasised tumours. The metastases showed decreased nuclear FGFR3 and membranous PTEN staining compared with corresponding primary tumours. EGFR expression was positively correlated with the expression of PTEN and FGFR3. The expression of ERBB2 was negatively correlated with p21RAS expression. According to our results, bladder carcinogenesis comprises FGFR3 translocation to the nucleus, upregulation of EGFR, ERBB2, KAI1 and PTEN; downregulation of p21RAS; and translocation of EGFR, ERBB2, and possibly PTEN to the membrane. Our results support the hypotheses regarding PTEN and KAI1 functioning as tumour suppressors in bladder cancer. EGFR and KAI1 may discriminate between nonmetastasised and metastasised cancers. A complex network of associations between the factors is suggested.
- PublicationOpen AccessRelevance of multidrug resistance 1 and P-glycoprotein to drug resistance in patients with systemic lupus erythematosus(Murcia : F. Hernández, 2007) Tsujimura, S.; Saito, K.; Nakayamada, S.; Tanaka, Y.Although corticosteroids and immunosuppressants are widely used for the treatments of various autoimmune diseases such as systemic lupus erythematosus (SLE), we often experience patients with SLE who are resistant to these treatments. Pglycoprotein (P-gp) of membrane transporters, a product of the multiple drug resistance (MDR)-1 gene, is known to play a pivotal role in the acquisition of drug resistance to chemotherapies in malignancy. However, the relevance of MDR-1 and P-gp to resting and activated lymphocyte, major targets of the treatments in autoimmune diseases, remains unclear. We found that peripheral lymphocytes in patients with SLE express Pgp on the surface and its expression is highly correlated with disease activity. P-gp on lymphocytes is induced by not only genotoxic stresses but also activation stimuli such as cytokines, resulting in active efflux of corticosteroids from cytoplasm of lymphocytes, resulting in drug-resistance and high disease activity. However, the addition of P-gp antagonists such as ciclosporin A and inhibitors of P-gp synthesis successfully reduce efflux of corticosteroids from lymphocytes in vitro and these results imply that P-gp antagonists and P-gp synthesis inhibitors could work in order to overcome drug-resistance in vivo. Therefore, we propose that the measurement of P-gp on lymphocytes is a useful marker to indicate drug resistance and requirement of antagonists and/or intensive treatments to overcome drug resistance in active SLE patients
- PublicationOpen AccessH-RAS gene expression in human multinodular goiter(Murcia : F. Hernández, 2007) Golbert, L.; Kolling, J.H.G.; Leitão, A.H.; Martins, L.; Kimura, E.T.; Maia, A.L.The RAS protooncogene has an important, although not yet established role in thyroid neoplasia. In this study, we evaluated the H-RAS mRNA and protein levels in human samples of nontoxic and toxic multinodular goiter samples, according to serum TSH levels. The mean of H-RAS mRNA levels in nodules of nontoxic nodular goiter were significantly increased compared to nonnodular tissue (1.49±1.21 vs. 0.94±0.81 AU, P=0.016). Nine of the 18 specimens (50%) of nontoxic multinodular goiter exhibited increased levels of H-RAS mRNA. The increased H-RAS mRNA levels were paralleled by inRAcreased H-Ras protein levels in about 90% of the cases. Interestingly, no differences were observed in H-RAS expression between nodules and adjacent nonnodular tissue in toxic nodular goiters (0.58±0.27 vs. 0.58±0.20 AU, P=0.88). None of the 10 samples from toxic multinodular goiters exhibited overexpression of H-RAS. The H-RAS expression was positively correlated with thyroglobulin expression (r2=0.51; P=0.04). In conclusion, we demonstrated increased levels of H-RAS mRNA and protein in samples of nontoxic multinodular goiter, indicating that it might be involved in goiter pathogenesis. In contrast, H-RAS overexpression was not detected in any of the samples of toxic multinodular goiter, suggesting different mechanisms for cell proliferation in nodular goiter according to thyroid status.
- PublicationOpen AccessDualism of mixed chimerism between hematopoiesis and stroma in chronic idiopathic myelofibrosis after allogeneic stem cell transplantation(Murcia : F. Hernández, 2007) Thiele, J.; Varus, E.; Siebolts, U.; Kvasnicka, H.M.; Wickenhauser, C.; Metz, K.A.; Beelen, D.W.; Ditschkowski, M.; Zander, A.; Kröger, N.Scant knowledge exists concerning lineagerestricted mixed chimerism (mCh) after allogeneic peripheral blood stem cell transplantation (PSCT) in patients with chronic idiopathic myelofibrosis (CIMF). Following a sex-mismatched PSCT, a combined immunopheno- and genotyping by fluorescence in-situ hybridization (FISH) was performed on sequential bone marrow (BM) biopsies at standardized intervals. Results were compared with PCR analysis of corresponding peripheral blood samples in five patients. According to FISH, pretransplant specimens revealed a gender congruence of more than 99%, while in the first three months the total BM exhibited a persistent fraction of host cells (30% to 40%) with a tendency to decline after about one year. It is noteworthy that the majority of endothelial cells maintained a recipient origin, whereas CD34+ progenitors and especially CD61+ megakaryocytes exhibited only very few host-derived cells. In keeping with the prevalence of donor cells in the hematopoietic compartment, PCR analysis of peripheral blood cells displayed a non-significant degree of mCh. In conclusion, according to FISH and PCR analysis, successful PSCT in CIMF results in an almost complete chimeric (donor-derived) state of the hematopoietic cell population. The non-transplantable stromal compartment includes the vascular endothelium with a predominance of recipient cells. The minimal mCh of this population implies probably a donor-derived origin (endothelial progenitor cells).
- PublicationOpen AccessHistopathological alterations, EROD activity, CYP1A protein and biliary metabolites in gilthead seabream Sparus aurata exposed to Benzo(a)pyrene(Murcia : F. Hernández, 2007) Ortiz-Delgado, J.B.; Segner, H.; Arellano, J.M.; Sarasquete, C.This study compared for seabream, Sparus aurata exposed to benzo(a)pyrene-B(a)P-, the response of molecular cytochrome P450 1A (CYP1A) and cellular histopathology biomarkers. Male gilthead seabream, Sparus aurata specimens were exposed for 20 days via water to a series of high B(a)P concentrations. CYP1A was assessed by measuring enzymatic activity (EROD) and CYP1A protein content, and cellular responses were evaluated by routine histopathological methods. In addition, biliary metabolites were measured in order to verify that B(a)P was absorbed and metabolised. Histological lesions, both in liver and gills, increased in parallel to B(a)P concentrations, with the majority of changes representing rather non-specific alterations. Hepatic EROD and CYP1A proteins data showed a concentration-dependent induction, while in the gills, EROD activity but not CYP1A proteins showed a nonmonotonous dose response, with a maximum induction level at 200 μ g B(a)P.L-1 and decreasing levels thereafter. The findings provide evidence that short-term, high dose exposure of fish can result in significant uptake and metabolism of the lipophilic B(a)P, and in pronounced pathological damage of absorptive epithelia and internal organs.
- PublicationOpen AccessA quantitative morphometric study of rectal mucosa in adult and aged healthy subjects(Murcia : F. Hernández, 2007) Milosevic, P.D.; Trbojevic, J.; Milicevic, N.M.; Bojic, D.; Davidovic, M.; Svorcan, P.; Dapcevic, B.; Bojic, B.; Mihajlovic, G.; Milicevic, Z.Rectal mucosa is relatively susceptible to pathological processes and frequently it is affected by various diseases. However, there is a notable lack of quantitative data regarding normal rectal mucosa, which would provide a reference for histoquantitative studies of the pathologically changed tissue. Therefore, we obtained the tissue from 27 healthy patients subjected to diagnostic rectoscopy during active screening for asymptomatic cancer of the large intestine, in which no disease was found. Using computer-aided morphometric analysis, we studied all structural elements of the rectal mucosa. The patients were divided into four groups according to the age and sex: adult males, elderly males, adult females and elderly females. The patients under 60 years of age were grouped as adult and those older than 60 years as aged subjects. A decreased height of surface epithelium was registered in both elderly male and female groups. This finding, however, was significant only when adult and elderly male groups were compared. The tendency towards reduction of the mucosal height was also registered comparing male adult and elderly groups. The number of crypts per 0.1 mm2 of tissue increased with aging in both males and females, whereby the crypts were always more numerous in males than in females. The increase in number of crypts in male subjects was accompanied by a decrease in their diameter and perimeter. The changes associated with ageing were discrete and affected only the male subjects.
- PublicationOpen AccessStructure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis(Murcia : F. Hernández, 2007) Xu, J.; Cheng, T.; Feng, H.T.; Pavlos, N.J.; Zheng, M.H.Excessive activity of osteoclasts becomes manifest in many common lytic bone disorders such as osteoporosis, Paget’s disease, bone aseptic loosening and tumor-induced bone destruction. Vacuolar proton pump H+-adenosine triphosphatases (V-ATPases), located on the bone-apposed plasma membrane of the osteoclast, are imperative for the function of osteoclasts, and thus are a potential molecular target for the development of novel anti-resorptive agents. To date, the V-ATPases core structure has been well modeled and consists of two distinct functional domains, the V1 (A, B1, B2, C1, C2, D, E1, E2, F, G1, G2, G3, and H subunits) and V0 (a1, a2, a3, a4, d1, d2, c, c’ e1, e2 subunits) as well as the accessory subunits ac45 and M8-9. However, the exact configuration of osteoclast specific V-ATPases remains to be established. Inactivation of subunit a3 leads to osteopetrosis in both mice and man because of nonfunctional osteoclasts that are capable of acidifying the extracellular resorption lacuna. On the other hand, inactivation of subunits c, d1 and ac45 results in early embryonic lethality, indicating that certain subunits, such as a3, are more specific to osteoclast function than others. In osteoclasts, V-ATPases also cooperate with chloride channel protein CLC-7 to acidify the resorption lacuna. In addition, development of V-ATPases inhibitors such as bafilomycin A1, SB 242784 and FR167356 that selectively target osteoclast specific VATPases remains a challenge. Understanding the subunits of V-ATPase regulate osteoclast function might facilitate the development of novel and selective inhibitors for the treatment of lytic bone disorders. This review summarizes recent research developments in VATPases with particular emphasis on osteoclast biology.
- PublicationOpen Access