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Immunohistochemistry
Kinds of Immunohistochemistry Terms modified by Immunohistochemistry Selected AbstractsExpression of Na+/HCO3, co-transporter proteins (NBCs) in rat and human skeletal muscleACTA PHYSIOLOGICA, Issue 1 2004J. M. Kristensen Abstract Aim:, Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results:, Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions:, Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2. [source] Oestrogen receptor , is expressed in adult human skeletal muscle both at the mRNA and protein levelACTA PHYSIOLOGICA, Issue 4 2003A. Wiik Abstract Aim:, There are two known oestrogen receptors (ER), oestrogen receptor , (ER,) and the recently cloned oestrogen receptor , (ER,). ER, mRNA has been detected in mouse, rat, bovine and human skeletal muscle. ER, mRNA has been detected in bovine skeletal muscle. To our knowledge, no study has investigated the expression of oestrogen receptor , in human skeletal muscle. Therefore, the primary aim of the present investigation was to study ER, mRNA and protein expression in human skeletal muscle. In addition the ER, expression was also studied. Methods:, Muscle biopsies were taken from vastus lateralis in six healthy adults (three women and three men). mRNA expression was detected with real-time PCR (TaqMan) and protein localization by immunohistochemistry. Results:, A clear expression of ER, and ER, mRNA was seen in skeletal muscle in all subjects. The ER, mRNA expression was 180 fold higher compared with that of ER, mRNA. Immunohistochemistry demonstrated positive staining for ER,, but not for ER,, with localization to the nuclei of skeletal muscle fibres. On average, 70% of all nuclei were ER, -positive. Conclusion:, The present study shows for the first time ER, mRNA and protein expression in human skeletal muscle tissue in both males and females. [source] Dystrophin upregulation in pressure-overloaded cardiac hypertrophy in ratsCYTOSKELETON, Issue 1 2003Masato Maeda Abstract Dystrophin is a cytoskeletal protein localized to the sarcolemma of skeletal and cardiac muscle, and neurons. We have recently demonstrated that a significant cardiac damage including myocytes injury, inflammation, and fibrosis, was found in dystrophin-deficient myocardium during pressure overload [Kamogawa et al., 2001: Cardiovasc Res 50:509,515]. However, little is known about how the cardiac sarcolemmal cytoskeleton produces qualitative and quantitative changes in response to pressure overload. Accordingly, we investigated dystrophin gene expression and protein accumulation during cardiac hypertrophy. Cardiac hypertrophy was produced by banding of the abdominal aorta of rats. Total RNA from the left ventricle of the heart was used for a quantitative reverse transcription-polymerase chain reaction (RT-PCR). Dystrophin mRNA expression significantly increased by 33 ± 18% at 1 day (P < 0.05) and 45 ± 19% at 2 days (P < 0.01) after banding, while G3PDH mRNA showed no significant change. RT-PCR for dystrophin tissue-specific exon 1 revealed that only muscle type promoter, but not non-muscle type promoter (brain and Purkinje-cell type), was activated immediately after banding. Immunohistochemistry for dystrophin showed intense cellular membrane staining with an increase in the perimeter of the myocytes by 14% at 3 days (46.3 ,m, P < 0.01) and 19% at 7 days (51.2 ,m, P < 0.01) after banding. Western blotting also showed dystrophin protein increased by 14 ± 6% at 2 days (P < 0.05) and by 32 ± 10% at 3 days (P < 0.01) after aortic banding. In conclusion, upregulation of dystrophin mRNA expression and protein accumulation occurs in response to cardiac hypertrophy. These data and the vulnerability of dystrophin-deficient myocardium to pressure overload suggest that dystrophin could play an important role in maintaining the integrity of the sarcolemma. Cell Motil. Cytoskeleton 55:26,35, 2003. © 2003 Wiley-Liss, Inc. [source] Eph,ephrin A system regulates murine blastocyst attachment and spreadingDEVELOPMENTAL DYNAMICS, Issue 12 2006Haruko Fujii Abstract Although numerous adhesion molecules are expressed on mammalian endometrial epithelial cells, there have not been any studies of a mechanism to prevent premature attachment of the embryo. In this study, we examined the possible involvement of Eph,ephrin interaction, which can induce repulsive forces. In mice, Eph A1, A2, and A4 were expressed on endometrial epithelial cells and ephrin A1,4 on blastocysts. Reverse transcriptase-polymerase chain reaction showed that mRNA expression of ephrin A1,4 on embryos transiently decreased around the implantation period. Immunohistochemistry demonstrated that the expression of Eph A1 on endometrial epithelial cells and ephrin A1 and A3 expression on embryos decreased at implantation sites. Recombinant Eph A1 reacted with cell the surface of ephrin A-bearing trophectoderm cells. Attachment assays using Eph A1-coated dishes showed that blastocyst attachment was reversibly inhibited by Eph A1. These findings suggest an important role of the Eph,ephrin A system in regulating the initial embryo,maternal contact during the cross-talk period that precedes embryo implantation. Developmental Dynamics 235:3250,3258, 2006. © 2006 Wiley-Liss, Inc. [source] Arrested differentiation and epithelial cell degeneration in zebrafish lens mutantsDEVELOPMENTAL DYNAMICS, Issue 4 2001Thomas S. Vihtelic Abstract In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities. © 2001 Wiley-Liss, Inc. [source] Development of otolith receptors in Japanese quailDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2010David Huss Abstract This study examined the morphological development of the otolith vestibular receptors in quail. Here, we describe epithelial growth, hair cell density, stereocilia polarization, and afferent nerve innervation during development. The otolith maculae epithelial areas increased exponentially throughout embryonic development reaching asymptotic values near posthatch day P7. Increases in hair cell density were dependent upon macular location; striolar hair cells developed first followed by hair cells in extrastriola regions. Stereocilia polarization was initiated early, with defining reversal zones forming at E8. Less than half of all immature hair cells observed had nonpolarized internal kinocilia with the remaining exhibiting planar polarity. Immunohistochemistry and neural tracing techniques were employed to examine the shape and location of the striolar regions. Initial innervation of the maculae was by small fibers with terminal growth cones at E6, followed by collateral branches with apparent bouton terminals at E8. Calyceal terminal formation began at E10; however, no mature calyces were observed until E12, when all fibers appeared to be dimorphs. Calyx afferents innervating only Type I hair cells did not develop until E14. Finally, the topographic organization of afferent macular innervation in the adult quail utricle was quantified. Calyx and dimorph afferents were primarily confined to the striolar regions, while bouton fibers were located in the extrastriola and Type II band. Calyx fibers were the least complex, followed by dimorph units. Bouton fibers had large innervation fields, with arborous branches and many terminal boutons. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 436,455, 2010 [source] Synchronous high-grade squamous intraepithelial lesion and adenocarcinoma in situ of cervix in a young woman presenting with hyperchromatic crowded groups in the cervical cytology specimen: Report of a caseDIAGNOSTIC CYTOPATHOLOGY, Issue 11 2008Nadeem Zafar M.D. Abstract We report a 29-year-old woman who underwent routine gynecologic evaluation at a community clinic and had a cervical sample drawn for liquid-based cytologic evaluation. At cytology, many hyperchromatic crowded groups (HCG) were present, but a consensus could not be established whether the abnormal cells were primarily glandular or squamous with secondary endocervical glandular involvement. An interpretation of atypical endocervical cells, favor neoplastic, was rendered and biopsy advised if clinically appropriate. At biopsy, the cervix contained synchronous squamous cell carcinoma in situ, secondarily involving endocervical glands, and neighboring adenocarcinoma in situ. Immunohistochemistry for Ki-67 and p16INK4A crisply and precisely stained both the lesions, clearly separating them from the adjacent uninvolved mucosa. This case re-emphasizes the challenge associated with accurate evaluation of HCG at cytology, the significance of ancillary testing for surrogate markers of high-risk HPV (HR-HPV) infection, the need for adjunct testing for HPV-DNA in the setting of HCG at cervical cytology, and a recommendation to set up studies to evaluate the role of surrogate markers of HR-HPV infection in cytologic samples with HCG. Diagn. Cytopathol. 2008;36:823,826. © 2008 Wiley-Liss, Inc. [source] Atypical papillary proliferation in gynecologic patients: A study of 32 pelvic washes,DIAGNOSTIC CYTOPATHOLOGY, Issue 2 2005Karyna C. Ventura M.D. Abstract Papillary clusters in gynecologic pelvic washes frequently cause diagnostic challenges because they can be associated with borderline or malignant ovarian tumors, as well as benign pelvic diseases. The objective of our study was to review all pelvic washes with atypical papillary proliferation (APP) and investigate whether cytomorphology and/or immunohistochemistry on cell block could determine their origin. Thirty-two pelvic washes from 31 patients containing APP were reviewed and correlated with their corresponding gynecologic or pelvic disease. Previously obtained cell blocks with immunohistochemical (IHC) stains were reviewed also. Nine of 32 washes (28%) were overcalled as malignant and were from patients with 5 borderline serous ovarian tumors (BSTO), 1 ovarian follicular cyst, 1 serous cystadenofibroma, and 1 endometrial carcinoma with ovarian seromucinous cystadenoma. BSTO and endometriosis were the most common sources of APP. Cell blocks could not discriminate further the etiology of APP. Immunohistochemistry was performed rarely and not fully contributory. Caution in interpreting papillary groups and cytohistological correlation is recommended to prevent a high false positive rate. Diagn. Cytopathol. 2005;32:76,81. © 2005 Wiley-Liss, Inc. [source] Hippocampal Malformations Do Not Necessarily Evolve into Hippocampal SclerosisEPILEPSIA, Issue 6 2005Arjune Sen Summary:,Purpose: Hippocampal malformations have been proposed to underlie or evolve into hippocampal sclerosis, a common cause of refractory partial epilepsy. We report two patients with chronic epilepsy and developmental abnormalities of the hippocampus and cortex. We seek to address, in patients with recurrent convulsive seizures over many decades, whether hippocampal malformations necessarily progress to hippocampal sclerosis. Methods: The first patient died at age 76 years and had experienced convulsive seizures for 43 years. The second patient, aged 64 years at death, had experienced convulsive seizures for 49 years. The brains were processed routinely. Immunohistochemistry for dynorphin and neuropeptide Y was performed. Results: The first case exhibited bilateral perisylvian polymicrogyria. Both hippocampi demonstrated abnormal convolution in the CA1 subfield and subiculum. In the second case, periventricular heterotopia was found in the wall of the right lateral ventricle. The right hippocampus was abnormally oriented with excessive convolutions of the pyramidal cell layer between CA1 and the subiculum. In neither patient did the hippocampi exhibit neuronal loss. Furthermore, dynorphin immunohistochemistry revealed no reactivity in the molecular layers, and staining with neuropeptide Y confirmed normal numbers of hilar interneurons. Conclusions: These two cases demonstrate histologically that, even in long-standing epilepsy, malformations of the hippocampus do not necessarily develop into hippocampal sclerosis. [source] NF-,B and apoptosis in colorectal tumourigenesisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2007M. M. Aranha Abstract Background, Nuclear factor-,B (NF-,B) may play an important role in colorectal tumourigenesis, controlling cell cycle and apoptosis gene expression. In addition, imbalances between cell proliferation and cell death are thought to underlie neoplastic development. The aims of this study were to investigate apoptosis and expression of several apoptosis-related proteins, and to determine correlations with colorectal tumour progression. Materials and methods, Apoptosis was evaluated by the TUNEL assay in 48 patient samples, including adenomas, adenocarcinomas and adjacent normal mucosas. Immunohistochemistry was performed for Bcl-2 and NF-,B. Expression levels of p53, Bax and I,B proteins were determined by immunoblotting. Cultured human colon cancer cells were used to evaluate NF-,B expression and nuclear translocation by immunocytochemistry and immunoblotting. Results, Apoptosis and NF-,B immunoreactivity were significantly higher in tumour tissue compared with normal mucosa (P < 0·01), increasing in association with histological tumour progression (P < 0·01). Bcl-2 was consistently higher in normal mucosa (P < 0·01) and inversely correlated with the percentage of apoptosis (P < 0·01). Phosphorylated p53 and Bax levels were similar in tumour tissue and normal mucosa; however, the NF-,B inhibitor, I,B, tended to decrease in tumours. In vitro, nuclear translocation of NF-,B was greater in proliferative than in resting phases of colon cancer cells. Conclusions, NF-,B expression and apoptosis are increased from adenoma to poorly differentiated adenocarcinoma tissues. Apoptosis is correlated with suppression of Bcl-2 expression, but appears to proceed through a p53- and Bax-independent pathway. Activation of NF-,B may play an important role in colorectal tumour progression. [source] Is my antibody-staining specific?EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008How to deal with pitfalls of immunohistochemistry Abstract Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto- and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false-positive results due to non-specific binding to tissue components or recognition of epitopes shared by several molecules. This ,Technical Spotlight' discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non-specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use. [source] Overexpression of APP provides neuroprotection in the absence of functional benefit following middle cerebral artery occlusion in ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Jared Clarke Abstract Cerebral ischaemia leads to a transient accumulation of ,-amyloid precursor protein (APP) and ,-amyloid (A,) peptides adjacent to the ischaemic lesion. There is conflicting evidence that APP/A, fragments may either enhance neuronal plasticity or be neurotoxic. The aim of the current study was to assess the effect of overexpression of human APP in rats on functional recovery following cerebral ischaemia. Adult APP-overexpressing (hAPP695 Tg) rats subjected to transient middle cerebral artery occlusion (MCAO) had significantly smaller infarct volumes than non-transgenic littermates, yet did not perform better on a series of sensorimotor or learning tests during a 6-month follow-up period. In fact, transgenic animals were found to be significantly more impaired in both the beam-walking and Morris water maze tests following MCAO. Immunohistochemistry showed human A,-positive staining in the cortex and hippocampus of APP transgenic rats. The present data suggest that while overexpression of APP in rats may provide some histological neuroprotection in the event of cerebral ischaemia, this does not translate into significant functional recovery. [source] Erythropoietin reduces Schwann cell TNF-,, Wallerian degeneration and pain-related behaviors after peripheral nerve injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2006W. Marie Campana Abstract Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-,). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-, has been implicated in the development of pain-related behaviors, we measured TNF-, mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-, mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-, in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-, immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-, in CCI by blocking expression of TNF-, in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-, expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-,in vitro. These results indicated that rhEpo regulates TNF-, by multiple mechanisms; rhEpo regulates TNF-, mRNA expression by Schwann cells but also may directly counteract TNF-, signaling pathways that lead to injury, chronic pain and/or death. [source] Tyrosine hydroxylase-positive neurons intrinsic to the human striatum express the transcription factor Nurr1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2004Martine Cossette Abstract The putative dopaminergic (DA) neurons intrinsic to human striatum were studied to determine their similarity with DA neurons of the substantia nigra pars compacta (SNpc). The comparison was based on morphological features and on the presence or absence of Nurr1, an orphan receptor of the nuclear receptor family that is essential for the expression of DA phenotype by developing SNpc neurons. Immunohistochemistry for the neuronal nuclear protein (NeuN; a neuronal marker) and in situ hybridization for tyrosine hydroxylase (TH) and/or Nurr1 were applied to post-mortem tissue obtained from seven normal individuals. On one hand, the TH-positive multipolar neurons in the human striatum, which were subdivided into three groups according to their size and pattern of dendritic arborization, were found to be morphologically similar to TH-positive neurons of the SNpc. The distribution frequency of striatal TH-positive neurons, according to their diameter, closely matches the frequency observed for multipolar TH-positive cells in the SNpc. On the other hand, the proportion of neurons expressing Nurr1 and TH mRNA transcripts on single striatal section was similar to the proportion of TH-immunoreactive neurons observed on adjacent sections. More importantly, in each striatum analysed, virtually all cells that stained for TH also expressed NeuN and Nurr1. This study provides novel data that confirm the existence of DA neurons intrinsic to the human striatum. It also provides the first evidence for the existence of striking morphological and chemical similarities between the DA neurons present at striatal level and those that populate the SNpc. [source] Decreased expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor in basal cell carcinomasEXPERIMENTAL DERMATOLOGY, Issue 11 2008Pierre E. Stoebner Abstract:, Thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor is associated with tumor angiogenesis. We evaluated the TP mRNA and protein expression in basal cell carcinomas (BCC) and in various skin tumors including numerous BCC histological simulants. Immunohistochemistry was performed on 99 paraffin sections of formalin-fixed skin tumors using monoclonal antibodies (mAb) against TP. TP mRNA levels were measured by real time RT-PCR in whole BCCs (wBCC) and laser capture microdissected (LCM) BCC tumor cells. TP immunostaining was negative in all BCC variants and in most of the benign trichogeneic tumors studied. By contrast, TP was constantly immunodetected in actinic keratosis (AK), squamous cell carcinomas (SCC), syringomatous carcinomas (SC), basosquamous carcinomas (BSC) and melanomas. TP mRNA levels were low and statistically not different in wBCC and normal skin but were strongly downregulated in LCM-BCC as compared with LCM-normal epidermis. We concluded that (i) anti-TP mAb is an useful marker to differentiate BCC from AK, SCC, BSC and SC but not from trichoblastic tumors, (ii) the lack of TP protein expression in BCC tumoral cells is linked to transcriptional regulatory mechanisms, (iii) the low TP mRNA levels in whole BCC may be related to the low intra-tumoral microvessel density, the slow growth and the very low metastatic potential of these tumors. [source] Immunohistochemistry and in situ hybridization in the study of human skin melanocytesEXPERIMENTAL DERMATOLOGY, Issue 3 2007Thierry Passeron Abstract: Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players as they produce and distribute melanin that protects the skin from ultraviolet (UV) radiation. In vitro experiments on melanocytic cell lines are useful to study melanogenesis and their progression towards melanoma. However, interactions of melanocytes with keratinocytes and with other types of cells in the skin, such as fibroblasts and Langerhans cells, are also crucial. We describe two techniques, immunohistochemistry (IHC) and tissue in situ hybridization (TISH), that can be used to identify and study melanocytes in the skin and their responses to UV or other stimuli in situ. We describe a practical method to localize melanocytic antigens on formalin-fixed, paraffin-embedded tissue sections and in frozen sections using indirect immunofluorescence with conjugated secondary antibodies. In addition, we detail the use of TISH and its combination with IHC to study mRNA levels of genes expressed in the skin at cellular resolution. This methodology, along with relevant tips and troubleshooting items, are important tools to identify and study melanocytes in the skin. [source] In vitro induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 expression in keratinocytes by boron and manganeseEXPERIMENTAL DERMATOLOGY, Issue 8 2004Nathalie Chebassier Abstract:, Matrix metalloproteinase (MMP)-2 and MMP-9 are involved in keratinocyte migration and granulation tissue remodeling during wound healing. Thermal water cures are sometimes proposed as complementary treatment for accelerating healing of wounds resulting from burns and/or surgery, but their mechanisms of action remain unknown. Some thermal waters are rich in trace elements such as boron and manganese. Interestingly, clinical studies have shown the beneficial effects of trace elements such as boron and manganese for human wound healing. To try to specify the role of trace elements in cutaneous healing, the present study investigated the effects of these trace elements on the production of MMP-2 and MMP-9 by normal human keratinocytes cultured in vitro. Immunohistochemistry and Western blot showed that intracellular MMP-9 expression in keratinocytes was induced when incubated for 6 h with boron at 10 µg/ml or manganese at 0.2 µg/ml. Moreover, gelatin zymography on keratinocyte supernatants showed an increase of gelatinase secretion after 24 h of incubation of keratinocytes with boron or manganese, regardless of concentration. Gelatinase secretion was not associated with keratinocyte proliferation induced by trace elements. Thus, our results suggest that boron and manganese could play a role in the clinical efficiency of thermal water on wound healing. [source] ,And the beat goes on' The cardiac conduction system: the wiring system of the heartEXPERIMENTAL PHYSIOLOGY, Issue 10 2009Mark R. Boyett The cardiac conduction system (CCS), consisting of the sino-atrial node, atrioventricular node and His,Purkinje system, is responsible for the initiation and co-ordination of the heart beat. In the last decade, our understanding of the CCS has been transformed. Immunohistochemistry, used in conjunction with anatomical techniques, has transformed our understanding of its anatomy; arguably, we now understand the position of the sino-atrial node (not the same as in medical textbooks), and our new understanding of the atrioventricular node anatomy means that we can compute its physiological and pathophysiological behaviour. Ion channel expression in the CCS has been shown to be fundamentally different from that in the working myocardium. Dysfunction of the CCS has previously been attributed to fibrosis, but it is now clear that remodelling of ion channels plays an important role in dysfunction during ageing, heart failure and atrial fibrillation. Differences in ion channel expression may even be responsible for the bradycardia in the athlete and differences in heart rate among different species (such as humans and mice). Recent work has highlighted less well-known components of the CCS, including tricuspid, mitral and aortic rings and even a third (retro-aortic) node. These additional tissues do not participate in the initiation and co-ordination of the heart beat and instead they are likely to be the source of various life-threatening arrhythmias. During embryological development, all parts of the CCS have been shown to develop from the primary myocardium of the linear heart tube, partly under the influence of the transcription factor, Tbx3. [source] Inhibition of PI3K/Akt partially leads to the inhibition of PrPC -induced drug resistance in gastric cancer cellsFEBS JOURNAL, Issue 3 2009Jie Liang Cellular prion protein (PrPC), a glycosyl-phosphatidylinositol-anchored membrane protein with unclear physiological function, was previous found to be upregulated in adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Overexpression of PrPC in gastric cancer has certain effects on drug accumulation through upregulation of P-glycoprotein (P-gp), which is suggested to play an important role in determining the sensitivity of tumor cells to chemotherapy and is linked to activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. In the present study, we further investigate the role of the PI3K/Akt pathway in PrPC -induced multidrug-resistance (MDR) in gastric cancer. Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrPC and phosphorylated Akt (p-Akt) expression in gastric cancer. Using established stable PrPC transfectant cell lines, we demonstrated that the level of p-Akt was increased in PrPC -transfected cells. Inhibition of PrPC expression by RNA interference resulted in decreased p-Akt expression. Inhibition of the PI3K/Akt pathway by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P-gp induced by PrPC. Taken together, our results suggest that PrPC -induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrPC -induced drug resistance and P-gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrPC regulates gastric cancer cell survival. [source] Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosaFEBS JOURNAL, Issue 24 2001Zhong Dong C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection. [source] Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coliGENES, CHROMOSOMES AND CANCER, Issue 2 2004Hendrik Bläker Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild-type allele. Inactivation of APC leads to impaired degradation of ,-catenin, thereby promoting continuous cell-cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ-line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second-hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of ,-catenin to the mutational status of the APC gene. Exon 3 of the ,-catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated ,-catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of ,-catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP-associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients. © 2004 Wiley-Liss, Inc. [source] Clinical significance of osteopontin expression in T1 and T2 tongue cancersHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 6 2008Chih-Yen Chien MD Abstract Background Osteopontin (OPN) is considered to be a tumor-related protein associated with tumor aggressiveness and metastasis. Methods Immunohistochemistry was used to study the clinical significance of OPN expression in T1 and T2 tongue cancers. Results Positive OPN expression significantly correlated with higher tumor classification (T) (p = .004), positive nodal classification (N) (p < .001), greater tumor thickness (p < .001), and presence of tumor necrosis (p = .016), respectively. The unfavorable cumulative 5-year disease-free survival rate significantly correlated with positive OPN expression (p < .001), T2 (p = .024), positive N (p < .001), greater tumor thickness (p = .023), and positive tumor necrosis (p = .003). However, taking CD105 into consideration, only CD105 expression was the independent prognostic factor for survival by Cox's regression analysis. Conclusion Overexpression of OPN in the tumors implicated a more aggressive tumor behavior and was an important factor for survival. In addition, there might be relationship between OPN and CD105 expressions in angiogenesis. © 2008 Wiley Periodicals, Inc. Head Neck, 2008 [source] Adult extracardiac rhabdomyoma: Light and immunohistochemical studies of two cases in the parapharyngeal spaceHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 3 2006Kristine Bjørndal Sørensen MD Abstract Background. We present two cases of adult rhabdomyoma in the parapharyngeal space. They are rare benign tumors with a characteristic histologic appearance. Methods. The tumors were studied by light and immunohistochemical analysis using stains characteristic of striated muscle fibers. Results. Cross-striation was demonstrated by phosphotungstic acid hematoxylin (PTAH), muscle specific actin, desmin, and myoglobin while dystrophin was expressed in the cell membranes. Clonal origin was confirmed by expression of myosin heavy chain-fast only. Expression of myosin-neonatal and myogenin proved slight proliferation with incipient differentiation in an otherwise mature tumor. Conclusion. The head and neck area harbors 90% of adult rhabdomyomas and should be considered in a differential diagnosis in this region. Immunohistochemistry confirms that the tumors are almost totally mature neoplasms of clonal origin. © 2006 Wiley Periodicals, Inc. Head Neck28: XXX,XXX, 2006 [source] Diagnosis and treatment of supraglottic laryngeal paraganglioma: report of a caseHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 1 2004John M. Del Gaudio MD Abstract Background. Paragangliomas of the larynx are unusual tumors that are seen as a vascular submucosal mass. These usually are seen in the supraglottic larynx but have also been found in the subglottis. This is the only laryngeal neuroendocrine tumor with a female predilection. It is important that paragangliomas be differentiated from other neuroendocrine tumors of the larynx, including atypical carcinoid, because of differing treatment modalities. Methods. We present the clinical, radiologic, and pathologic findings of a supraglottic laryngeal paraganglioma seen in a 50-year-old woman with a 6-month history of slowly progressive hoarseness. Results. The tumor was successfully approached by means of a midline laryngofissure with mucosal preservation. The patient remains disease free 24 months after surgery. Conclusions. Preoperative CT and angiography are useful in making the diagnosis of paraganglioma before surgical intervention. Complete excision through an external mucosa-sparing approach is the treatment of choice. Distinguishing laryngeal paraganglioma from other neuroendocrine tumors can be difficult. Immunohistochemistry is an important tool for the correct pathologic diagnosis. © 2004 Wiley Periodicals, Inc. Head Neck26: 94,98, 2004 [source] Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis,HEPATOLOGY, Issue 6 2009Jui-Chu Yang Ground glass hepatocytes (GGH) in chronic hepatitis B virus (HBV) infection harbor HBV pre-S deletion mutants in endoplasmic reticulum (ER) and exhibit complex biologic features such as ER stress, DNA damage, and growth advantage. The presence of pre-S mutants in serum has been shown to predict the development of hepatocellular carcinoma (HCC) in HBV carriers. GGHs hence represent a potentially preneoplastic lesion. Whether a specific growth factor is overexpressed and activated in GGHs remains to be clarified. In this study, growth factor(s) up-regulated by pre-S mutants was identified using a growth factor array in HuH-7 cells. Immunohistochemistry, reverse-transcriptase polymerase chain reaction, and Western blot analysis were performed to study the participation of these genes and their signal pathways in HuH-7 cells and liver tissues. We demonstrate that vascular endothelial growth factor-A (VEGF-A) was up-regulated by pre-S mutants in HuH-7 cells and further confirmed in GGHs by immunostaining. The VEGF-A up-regulation by pre-S mutants could be suppressed by vomitoxin, an ER stress inhibitor. Furthermore, pre-S mutants-expressed HuH-7 cells exhibited activation of Akt/mTOR (mammalian target of rapamycin) signaling and increased growth advantage, which could be inhibited by VEGF-A neutralization. Consistent with this notion, enhanced expression of VEGF-A and activation of Akt/mTOR signaling, comparable to the levels of paired HCC tissues, were also detected in HBV-related nontumorous livers. Conclusion: The enhanced expression of VEGF-A in GGHs provides potential mechanism to explain the progression from preneoplastic GGHs to HCC in chronic HBV infection. (HEPATOLOGY 2009;49:1962,1971.) [source] Proteomic profiling reveals the prognostic value of adenomatous polyposis coli,end-binding protein 1 in hepatocellular carcinoma,HEPATOLOGY, Issue 6 2008Tatsuya Orimo Histological differentiation is a major pathological parameter associated with poor prognosis in patients with hepatocellular carcinoma (HCC) and the molecular signature underlying HCC differentiation may involve key proteins potentially affecting the malignant characters of HCC. To develop prognostic biomarkers for HCC, we examined the global protein expression profiles of 45 surgically resected tissues, including 27 HCCs with different degree of histological differentiation, 11 adjacent nontumor tissues, and seven normal liver tissues. Unsupervised classification grouped the 45 samples according to their histological classification based on the protein expression profiles created by laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE). Statistical analysis and mass spectrometry identified 26 proteins with differential expression, of which 14 were functionally linked to c-Myc, AP-1, HIF1A, hepatocyte nuclear factor 4 alpha, or the Ras superfamily (RhoA, CDC42, and Rac1). Among the proteins identified, we focused on APC-binding protein EB1 (EB1) because it was dominantly expressed in poorly differentiated HCCs, which generally correlate with the poor prognosis in patients with HCC. In addition, EB1 is controlled by c-Myc, RhoA, and CDC42, which have all been linked to HCC malignancy. Immunohistochemistry in a further 145 HCC cases revealed that EB1 significantly correlated with the degree of histological differentiation (P < 0.001), and univariate and multivariate analyses indicated that EB1 is an independent prognostic factor for recurrence (hazard ratio, 2.740; 95% confidence interval, 1.771,4.239; P < 0.001) and survival (hazard ratio, 2.256; 95% confidence interval, 1.337,3.807; P = 0.002) of patients with HCC after curative surgery. Conclusion: Proteomic profiling revealed the molecular signature behind the progression of HCC, and the prognostic value of EB1 in HCC. (HEPATOLOGY 2008;48:1851-1863.) [source] Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosisHEPATOLOGY, Issue 2 2000Nils Kinnman M.D. The authors examined the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and its relationship to histopathological changes in cystic fibrosis (CF) liver tissue. Immunohistochemistry was used to examine expression of CFTR, intercellular adhesion molecule-1 (ICAM-1) and liver cell-type markers in liver cryosections in 11 patients with CF-associated liver disease, and non-CF controls with (n = 17) and without (n = 3) liver disease. In CF patients prominent inflammatory infiltrates were not found, yet hepatic stellate cells were identified within fibrotic areas around bile ducts. Proliferating bile ducts displayed ICAM-1 immunoreactivity in 3 cases, but bile ducts were otherwise negative. In 2 patients homozygous for R764X and for 1112delT no CFTR immunoreactivity was detected. Bile-duct epithelial cells in patients carrying the ,F508 mutation displayed aberrant cytoplasmic immunolocalization of CFTR, as determined with confocal laser scanning microscopy, in contrast to the distinct CFTR expression at the luminal surface seen in controls. No clear relationship between CFTR expression and fibrosis or inflammation was evidenced in CF patients. In conclusion, these findings are consistent with an impairment of ,F508 CFTR processing in intrahepatic biliary epithelium. ICAM-1 expression on bile-duct epithelial cells and inflammatory infiltrates were rare findings in CF liver tissue, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease. [source] Alcohol inhibition of neurogenesis: A mechanism of hippocampal neurodegeneration in an adolescent alcohol abuse modelHIPPOCAMPUS, Issue 5 2010Stephanie A. Morris Abstract Adolescents diagnosed with an alcohol use disorder show neurodegeneration in the hippocampus, a region important for learning, memory, and mood regulation. This study examines a potential mechanism by which excessive alcohol intake, characteristic of an alcohol use disorder, produces neurodegeneration. As hippocampal neural stem cells underlie ongoing neurogenesis, a phenomenon that contributes to hippocampal structure and function, we investigated aspects of cell death and cell birth in an adolescent rat model of an alcohol use disorder. Immunohistochemistry of various markers along with Bromo-deoxy-Uridine (BrdU) injections were used to examine different aspects of neurogenesis. After 4 days of binge alcohol exposure, neurogenesis was decreased by 33 and 28% at 0 and 2 days after the last dose according to doublecortin expression. To determine whether this decrease in neurogenesis was due to effects on neural stem cell proliferation, quantification of BrdU-labeled cells revealed a 21% decrease in the dentate gyrus of alcohol-exposed brains. Cell survival and phenotype of BrdU-labeled cells were assessed 28 days after alcohol exposure and revealed a significant, 50% decrease in the number of surviving cells in the alcohol-exposed group. Reduced survival was supported by significant increases in the number of pyknotic-, FluoroJade B positive-, and TUNEL-positive cells. However, so few cells were TUNEL-positive that cell death is likely necrotic in this model. Although alcohol decreased the number of newborn cells, it did not affect the percentage of cells that matured into neurons (differentiation). Thus, our data support that in a model of an adolescent alcohol use disorder, neurogenesis is impaired by two mechanisms: alcohol-inhibition of neural stem cell proliferation and alcohol effects on new cell survival. Remarkably, alcohol inhibition of neurogenesis may outweigh the few dying cells per section, which implies that alcohol inhibition of neurogenesis contributes to hippocampal neurodegeneration in alcohol use disorders. © 2009 Wiley-Liss, Inc. [source] Induction of the vascular endothelial growth factor pathway in the brain of adults with fatal falciparum malaria is a non-specific response to severe diseaseHISTOPATHOLOGY, Issue 2 2010Isabelle M. Medana Medana I M, Day N P J, Roberts R, Sachanonta N, Turley H, Pongponratn E, Hien T T, White N J. & Turner G D H (2010) Histopathology,57, 282,294 Induction of the vascular endothelial growth factor pathway in the brain of adults with fatal falciparum malaria is a non-specific response to severe disease Aims:, Pathological or neuroprotective mechanisms in the brain in severe malaria may arise from microvascular obstruction with malaria-parasitized erythrocytes. This study aimed to investigate the role of hypoxia and induction of the vascular endothelial growth factor (VEGF) pathway in the neuropathophysiology of severe malaria. Methods and results:, Immunohistochemistry was performed on post mortem brain tissue sections from 20 cases of severe malaria and examined for the expression of transcriptional regulators of VEGF [hypoxia-inducible factor-1 alpha (HIF-1,), HIF-2,], DEC-1, VEGF, VEGF receptors 1 and 2, and the activated, phosphorylated VEGF receptor 2 (pKDR). HIFs showed limited protein expression and/or translocation to cell nuclei in severe malaria, but DEC-1, which is more stable and regulated by HIF-1,, was observed. There was heterogeneous expression of VEGF and its receptors in severe malaria and non-malarial disease controls. pKDR expression on vessels was greater in malaria cases than in controls but did not correlate with parasite sequestration. VEGF uptake by malaria parasites was observed. Conclusions:, VEGF and its receptor expression levels in severe malaria reflect a non-specific response to severe systemic disease. Potential manipulation of events at the vasculature by the parasite requires further investigation. [source] Assessment of a HER2 scoring system for gastric cancer: results from a validation studyHISTOPATHOLOGY, Issue 7 2008M Hofmann Aims:, Human epidermal growth factor receptor 2 (HER2) overexpression/amplification is implicated in the development of various solid tumour types. Validated methods and scoring systems for evaluating HER2 status exist in breast cancer, but not in gastric cancer. The aim was to establish a HER2 scoring system for gastric cancer to identify suitable patients for enrolment in a trial of trastuzumab (Herceptin®) in advanced metastatic gastric cancer. Methods and results:, Formalin-fixed paraffin-embedded gastric cancer samples were tested for HER2 status using the fluorescence in situ hybridization (FISH) pharmDxÔ kit (Dako Denmark A/S). Immunohistochemistry (IHC) was performed using the HercepTestÔ (Dako). Concordance between FISH and IHC was 93.5% in 168 evaluable samples. Eleven samples were scored as FISH+ but IHC, or equivocal. Conclusions:, IHC/FISH discrepancies were attributed to basolateral membranous immunoreactivity of glandular cells resulting in incomplete membranous reactivity and/or a higher rate of tumour heterogeneity in gastric cancer compared with breast cancer. With modifications to the IHC scoring system, the HercepTestÔ is considered valid for the identification of HER2+ gastric tumours for this clinical trial. Correlation of HER2 scores with clinical outcomes will be needed to determine which patients might benefit from trastuzumab therapy. [source] | |||||||||||||||||||||||||||||||||||||||||||