Histology and histopathology Vol.26, nº7 (2011)
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- PublicationOpen AccessThe function of TRPS1 in the development and differentiation of bone, kidney, and hair follicles(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Gai, Zhibo; Gui, Ting; Muragaki, YasuteruTRPS1 is a gene involved in Tricho-rhino-phalangeal syndrome (TRPS), an autosomal dominant skeletal disorder. TRPS1 encodes a GATA-type transcription factor that has nine zinc-finger motifs. A variety of mutations in TRPS1 including deletions and insertions, have been found in patients with TRPS type I and III. The functions of each domain of TRPS1 have been clarified from study of these mutations. Further studies on the localization and the function of TRPS1 have been performed using TRPS1Δgt and Trps1-deficient mice, which allow examination of the development and differentiation of all tissues with Trps1 expression. These studies suggest that TRPS1 exhibits a variety of functions in cartilage, kidneys, and hair follicles. In the growth plate cartilage, TRPS1 regulates the differentiation, proliferation, and apoptosis of chondrocytes through interaction of several signaling molecules. In addition, TRPS1 has a function downstream of BMP7, which regulates the mesenchymal-epithelial transition when nephrons are formed in renal development. Furthermore, TRPS1 suppresses the epithelial-mesenchymal transition and renal fibrosis induced by unilateral ureteral obstruction by decreasing Arkadia expression. Finally, TRPS1 is expressed in the dermal papillae and the mesenchymal cells surrounding the hair pegs, and the loss of TRPS1 largely influences the development of hair follicles. The molecular mechanisms of the function of TRPS1 in cartilage, kidneys, and hair follicles are discussed in this review.
- PublicationOpen AccessCell- and gene-therapy approaches to inner ear repair(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Conde de Felipe, M.M.; Feijoo Redondo, A.; García-Sancho, J.; Schimmang, T.; Durán Alonso, M.B.Sensorineural hearing loss is the most common sensory disorder in humans. It is primarily due to the degeneration of highly specialised mechanosensory cells in the cochlea, the so-called hair cells. Hearing problems can also be caused or further aggravated by the death of auditory sensory neurons that convey the information from the hair cells to the brain stem. Despite the discovery of stem/progenitor cells in the mammalian cochlea, no regeneration of either damaged hair cells or auditory neurons has been observed in mammals, in contrast to what is seen in avians and non-mammalian vertebrates. The reasons for this divergence have not yet been elucidated, although loss of stem cells and/or loss of their phenotypic plasticity in adult mammals have been put forward as possible explanations. Given the high incidence of this disorder and its economic and social implications, a considerable number of research lines have been set up aimed towards the regeneration of cochlear sensory cell types. This review summarizes the various routes that have been explored, ranging from the genetic modification of endogenous cells remaining in the inner ear in order to promote their transdifferentiation, to the implantation of exogenous stem or progenitor cells and their subsequent differentiation within the host tissue. Prophylactic treatments to fight against progressive sensory cell degeneration in the inner ear are also discussed.
- PublicationOpen AccessProspective evaluation of the learning curve of confocal laser endomicroscopy in patients with IBD(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Neumann, Helmut; Vieth, Michael; Atreya, Raja; Neurath, Markus F.; Mudter, JonasBackground and aims: Confocal laser endomicroscopy (CLE) represents a novel endoscopic imaging technique which enables the in vivo microscopic imaging within the mucosal layer of the gut at subcellular resolution. Currently, there are no data available on the learning curve of CLE, which was therefore the aim of our study. Methods: Twenty-six consecutive patients with inflammatory bowel disease (IBD) underwent total colonoscopy and were examined by fluorescein-aided CLE. Image data were collected and reviewed by two endoscopists in a blinded fashion. CLE images were compared to endoscopical and histological findings. Prospectively, the following performance parameters were documented: total duration of the procedure, confocal imaging time, time to receive a confocal image in focus, number of confocal images, number of confocal images in focus, CLE diagnosis and final histopathological diagnosis. Results: A significance decrease of CLE duration was detected between the first 8 and the subsequent cases (p=0.002). Confocal imaging time and the time to receive an image in focus declined significantly over time (p=0.0001), while number of images in focus significantly increased (p=0.0007). Agreement between CLE and histopathology improved over time with kappa values of 0.81 after twenty-six cases. Conclusions: There was a significant improvement in CLE performance over time, including decreased confocal imaging time, successful CLE diagnosis and decline in procedural time. These parameters improved significantly after the initial three cases. Therefore, CLE represents an easy to learn and apply novel diagnostic method for in vivo analysis and diagnosis in IBD.
- PublicationOpen AccessBiological effects of low-frequency pulsed magnetic fields on the embryonic central nervous system development. A histological and histochemical study(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Roda, Olga; Garzón, Ingrid; Carriel, Víctor; Alaminos, Miguel; Sánchez-Montesinos, IndalecioNumerous experiments have yielded contradictory results on the harmful action of magnetic fields on embryonic development. Pulsed magnetic fields appear to be able to delay normal development of embryos. In the present study, fertilized Gallus domesticus eggs were exposed during incubation to pulsed magnetic fields (harmonic signals of 10 µT for 1 second with silences of 0.5 seconds) of 50 or 100 Hz frequency. Embryos extracted at 45 h of exposure to fields of 50 Hz or 100 Hz frequency had significantly (p<0.05) fewer somite pairs compared with controls of the same age. At 15 days of incubation, only embryos exposed to a 10 µT- 50 Hz field had a significantly (p<0.05) higher somatic weight. At 21 days of incubation, a significantly lower somatic weight (p<0.01) and development stage (p<0.05) was found in embryos exposed to a 10 µT-100 Hz field than in controls, while a lower development stage (p<0.05) alone was observed in those exposed to a 10 µT-50 Hz field. In addition, animals showed higher expression of the neural marker NSE (neural specific enolase) after 21 days of development as determined by immunohistochemistry, with very low expression of glycosaminoglycans identified by alcyan blue staining. These results suggest that pulsed magnetic fields may be able to hinder normal embryonic development in vivo and to alter normal neural function, at least at the intensities and frequencies analyzed in the present study
- PublicationOpen AccessDifferent patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Santin, G.; Piccolini, V.M.; Veneroni, P.; Barni, Sergio; Bernocchi, G.; Bottone, M.G.Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 µM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspaseindependent apoptotic pathways in B50 rat neuronal cells.
- PublicationOpen AccessExpression and regulation of nicotine receptor and osteopontin isoforms in human pancreatic ductal adenocarcinoma(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Sullivan, Jennifer; Blair, Laurel; Alnajar, Amer; Aziz, Tamer; Chipitsyna, Galina; Gong, Qiaoke; Yeo, Charles J.; Arafat, Hwyda A.Osteopontin (OPN) is a secreted phospho-protein that confers on cancer cells a migratory phenotype. We have recently shown that nicotine, a risk factor in pancreatic ductal adenocarcinoma (PDA), induces an alpha7-nicotine acetylcholine receptor (α7-nAChR)-mediated increase of OPN in PDA cells. In this study, we tested nicotine’s effect on the expression of OPN splice variants (OPNa, b, c) in PDA cells. We also analyzed the correlation between patients’ smoking history with OPN and α7-nAChR levels. RT-PCR and UV-light-illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and PDA cells treated with or without nicotine (3-300 nM). Localization of total OPN, OPNc and α7-nAChR was analyzed by immunohistochemistry, and their mRNA tissue expression levels were correlated with the patients’ smoking history. PDA cells expressed varying levels of OPNa, OPNb, and α7-nAChR. Nicotine treatment selectively induced denovo expression of OPNc and increased α7-nAChR expression levels. In PDA tissue, OPNc was found in 87% of lesions, of which 73% were smokers. OPNc and total OPN levels were correlated in the tissue from patients with invasive PDA. Nicotine receptor was expressed in the invasive and premalignant lesions without clear correlation with smoking history. We show here for the first time that α7-nAChR is expressed in PDA cells and tissues and is regulated by nicotine in PDA cells. This, together with our previous findings that α7-nAChR mediates the metastatic effects of nicotine in PDA, suggest that combined targeting of α7-nAChR and OPNc could be a valid novel therapeutic strategy for invasive PDA, especially in the smoking population.
- PublicationOpen Accessß-Lapachone accelerates the recovery of burn-wound skin(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Fu, Shih-Chen; Chau, Yat-Pang; Lu, Kuo-Shyan; Kung, Hsiu-Niß-lapachone is a quinone of lapachol extracted from the bark of lapacho tree. Recent findings demonstrated that punched skin wounds of mice healed faster with ß-lapachone treatment. The present study investigates the effects of ß-lapachone on burn-wound skin of C57BL/6 mice injured by a 100°C iron stick. Our results indicated that wounds treated with ß-lapachone recovered faster than those treated with control ointment containing no ß-lapachone. On the third day after burning, the area of ß-lapachone treated-wound was 30% smaller than wound treated with control ointment. H&E and immunohistochemistry staining showed that burn-wound skin treated with ointment containing ß-lapachone healed faster in its epidermis, dermis, and underlying connective tissues with more macrophages appeared than those treated with control ointment alone. RAW264.7 cell, a macrophage-like cell line derived from BALB/C mice, was used as a model for scrutinizing the effect of ß-lapachone on macrophages. We found that the proliferation and the secretion of EGF and VEGF by macrophages were higher in cultures treated with ß-lapachone and that ß-lapachone can also increase the release of EGF with TNF-α pretreatment. We conclude that ß-lapachone plays an important role in accelerating burn wound healing, and that ß-lapachone not only can raise the proliferation of macrophages but also increase the release of VEGF from macrophages.
- PublicationOpen AccessHistopathological changes in gerbil liver and kidney after aluminium subchronic intoxication(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Garrosa, Manuel; Llanes, Felipe; Gayoso, Manuel J.Young gerbil livers and kidneys were analyzed by means of light and electron microscope to assess the histopathological changes caused by prolonged systemic aluminum (Al) administration. The experimental group was injected with AlCl3 i.p. for 5 weeks, while litter mates received PBS as sham-injected controls or served as untouched controls. Mortality occurred in 33% of experimental and 12.5% of sham-injected groups. The animals were perfused intracardially with 1% glutaraldehyde plus 1% paraformaldehyde and samples of liver and kidneys were processed for aluminum and iron histochemistry and conventional light- and transmission electron microscopy. White deposits composed of cellular debris appeared on the surface of liver and kidneys and in the mesentery as a consequence of Al treatment. Adherences of Glisson capsule to the diaphragm, as well as scattered small foci of hepatocyte necrosis with non-caseificant microgranulomas and mild portal inflammation, developed in the experimental group. Sham-injected animals also exhibited these granulomas but to a lesser degree. Al deposits were found in experimental animal granulomas and inside macrophages cytoplasm scattered throughout the liver. Iron deposition appeared in pericentral hepatocytes of experimental animals, in granulomas and in portal spaces of the three groups of animals. Ultrastructurally, hepatocytes of experimental animals showed mitochondria hyalinization, disintegration of endoplasmic reticulum and clustering of ribosomes. Phagolysosomes appeared larger and occurred more frequently in both hepatocytes and Kupffer cells of experimental animals. In 2 out of the 6 experimental animals studied, tubular atrophy was present in the renal cortical region, the kidneys of the remaining animals appearing normal. Al and iron were found very occasionally in the kidney parenchyma of experimental animals, while isolated mesangial cells showed iron deposits in a few glomeruli of both experimental and the two groups of control animals.
- PublicationOpen AccessImmunohistochemical study of the apoptosis process in epidermal epithelial cells of rats under a physiological condition(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Udayanga, K.G.S; Miyata, H.; Yokoo, Y.; Qi, W.M.; Takahara, E.; Mantani, Y.; Yokoyama, T.; Hoshi, N.; Kitagawa, HiroshiEpidermal homeostasis is maintained by both epithelial proliferation in the stratum basale (SB) and the apoptosis of epithelial cells under physiological conditions. In this study, the induction and regulation mechanisms of epidermal apoptosis were immunohistochemically investigated in the epidermis from Wistar rat’s palm and foot pad by using several apoptotic related proteins under a physiological condition. The results showed that Fas and Fas-L were expressed in cellular membranes of the stratum spinosum (SS), whereas TNF-R1 did not show any membranous expression in any epidermal layers. TNF-α was not observed in the epidermis. Caspase-10, cleaved caspase-3 and DNase-1 were found in the epithelial cytoplasms from the SS to stratum granulosum (SG), whereas caspase-8 was not detected in the epidermis. XIAP and Bak were found in the cytoplasm from the SS to SG, and the intensity of Bak-positivity was stronger in the SG than the SS, whereas Bid, Apaf-1 and cleaved caspase-9 were restricted in the SG. Homogenous cytoplasmic immunoreactivity of Bcl-2 was found in the SB and the intensity was gradually decreased from the SB to the SG. The granular-cytoplasmic immunopositivity of cytochrome C gradually altered into homogenous cytoplasmic expression in the upper half of the SG. Single-stranded DNA was rarely detected in the upper portion of the SG. These results suggest that epidermal apoptosis is induced by the interaction between Fas and Fas-L and the activation of caspase-10, and might initially proceed through a mitochondrialindependent pathway, and that a mitochondrialdependent pathway finally accelerated under physiological conditions.
- PublicationOpen AccessSignificance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Ina, Keisuke; Kitamura, Hirokazu; Tatsukawa, Shuji; Fujikura, YoshihisaMyofibroblast transdifferentiation plays a crucial role in the development and progression of renal tubulointerstitial fibrosis. However, the significance of α-smooth muscle actin (α-SMA) expression, which is the major morphological characteristic of myofibroblasts, remains to be determined in detail. The effect of α-SMA expression on fibrosis tissue was examined by using a fibrosis model (collagen gel) in vitro. The transdifferentiation of fibroblasts into myofibroblasts was triggered in the culture medium with 0.5% fetal bovine serum (FBS)+transforming growth factor (TGF)- ß1, but not with 10% FBS+TGF-ß1. The TGF-ß1- induced gel contraction caused by myofibroblasts was greater than that by fibroblasts. Gel contraction by myofibroblasts involved the Ca2+-dependent myosin light chain kinase pathway, as well as the activation of Rho kinase and p38 mitogen-activated protein kinase (MAPK). Taken together, these findings suggest that α- SMA expression in renal interstitial fibroblasts, i.e., myofibroblast transdifferentiation, accelerates the contraction of the tubulointerstitial fibrosis tissue via the Ca2+-dependent pathway, in addition to the pathways involved in fibroblast contraction; this event may lead to renal atrophy and renal failure.
- PublicationOpen AccessComparative immunohistochemical study of tissue integration of macroporous and laminar surgical meshes(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Takács, Ildikó; Horváth, Szabolcs; Molnár, Ágnes; Gáspár, Sarolta; Hajós, Rebeka; Meczker, Ágnes; Kóbor, Péter; Lantos, János; Jávor, Szaniszló; Balatonyi, Borbála; Szekeres, György; Rőth, Erzsébet; Wéber, GyörgyIntraperitoneal surgical mesh implantation is required for laparoscopic ventral hernia repair. Composite meshes are well known in animal models and human practice. The aim of our study is to compare the biological behaviour of two different textured siliconecovered polypropylene meshes. Transmural abdominal wall defect was created in 40 rabbits and treated as follows: In 20 animals a polypropylene mesh with a laminar silicone covering (LSPP) and in the rest a macroporous textured mesh knitted of silicone-impregnated polypropylene filaments (MSPP) was applied. One and three weeks after implantation we evaluated the intraperitoneal adhesion formation of the mesh macroscopically, histologically and immunohistochemically to detect the reactive cells, especially inflammatory, endothelial and mesothelial cells, as well as their proliferative activity, and with Scanning Electron microscopy to visualize the surface of the meshes. The adhesion formation caused by the composites showed no statistical difference after one week although in the three weeks old samples the LSPP adhesion was significantly weaker than that of MSPP. As complications, serome formation in both groups, fistulas, abscesses, and sc. haematoma in the LSPP group were found. Only in MSPP containing tissues was the decrease of Ki-67 positive proliferating cells significant. A significant increase in VEGF expressing cells was observed only in MSPP containing three week old samples, suggesting better regulation of vascular growth in tissues surrounding the implants. In one week old specimens we observed an irregular proliferation of cytokeratin containing mesothelial cells in both group. The intraperitoneal surface of MSPP mesh was covered with neoperitoneum, while it was not regurarly seen on LSPP mesh after three week.
- PublicationOpen AccessImmunosuppressive cells and tumour microenvironment: Focus on mesenchymal stem cells and myeloid derived suppressor cells(F. Hernández y J.F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología., 2011) Bianchi, Giovanna; Borgonovo, Giacomo; Pistoia, Vito; Raffaghello, LizziaTumours have been compared to unhealed wounds that produce large amounts of inflammatory mediators, including cytokines, chemokines, and growth factors. These molecules participate in the formation of a rich and heterogeneous microenvironment by attracting non malignant cells that promote tumour progression and dissemination. Tumour infiltrating cells include macrophages, myeloid-derived suppressor cells (MDSCs), mesenchymal stromal cells (MSCs) and TIE2-expressing monocytes. Most of them are bone marrow-derived, although MSC are present in virtually every tissue. This review focuses on MDSCs and MSCs, both of which can exert pro-tumorigenic effects through negative regulation of immune responses. MDSCs represent a heterogeneous population of cells of myeloid origin that are expanded and activated in response to growth factors and cytokines released by tumours. Once MDSCs are activated, they accumulate in lymphoid organs and tumours where they exert T cell immunosuppression. Like MDSCs, MSCs can be mobilized from the bone marrow into the bloodstream and home in the tumour stroma, where they either help or hinder tumour growth. Here, we will discuss the origin, the functions and the mechanisms of action of MSCs and MDSCs, as well as the strategies to target these cells for the therapeutic benefit of cancer patients.
- PublicationOpen AccessC-Reactive protein and embolization during carotid artery stenting. A serological and morphological study(F. Hernández y Juan F. Madrid. Murcia: Universidad de Murcia, Departamento de Biología Celular e Histología, 2011) Faggioli, G.; Fittipaldi, S.; Pini, R.; Beltrandi, E.; Mauro, R.; Freyrie, A.; Rapezzi, C.; Stella, A.; Pasquinelli, G.Introduction. High-sensitivity C-Reactive Protein (hsCRP) levels are correlated with vulnerable carotid plaques, although their impact on the outcome of carotid revascularization is unknown. The aim of our study was to investigate the correlation between hsCRP and embolization during carotid artery stenting (CAS). Methods. Patients with symptomatic carotid stenosis were submitted to CAS with distal protection filters. Serum hsCRP was analysed prior to CAS and patients were divided into two groups: Class I, patients presenting hsCRP<5 mg/l and, Class II, patients presenting hsCRP≥5 mg/l. Plaques were categorised by ultrasound grey scale measurement as homogenous and dishomogenous. Afterwards CAS filters were analyzed microscopically and ultrastructurally to determine the type and the amount of debris present, based on percentage of surface involvement (SI) and pore occluded (PO) by embolic material. Results. Fourteen patients underwent uneventful CAS, with a mean hsCRP of 11.5±18.4 mg/l. Eight patients were in Class I and six in Class II. All filters had microscopic debris. SI was 25.4% in Class I and 33.3% in Class II (p=ns), PO 22.9% and 33.3% respectively (p=0.049). Patients in Class II who also had a dishomogenous plaque showed greater SI and PO compared with patients in Class I with homogenous plaque (35.0% vs. 21.8% and 40.4% vs. 22.7% respectively, p<0.05). Microscopically embolic material was identified as atherosclerotic plaque fragments and platelet aggregates and was similar in both groups. Discussion. High hsCRP levels are associated with significantly greater embolization during CAS in symptomatic patients, particularly in dishomogenous plaque. Although these results need further investigation due to the limited number of enrolled patients, this study suggests that CAS may not be indicated as a method of carotid revascularization in this setting.