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Immunocytochemical Detection (immunocytochemical + detection)
Selected AbstractsImmunocytochemical Detection of Synaptophysin in Enteric Neurones during Prenatal Development in the Rat StomachANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2004M. Asar Summary In this study, the localization and appearance of synaptophysin-immunoreactive (IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry and light microscopy in the embryonic rat stomach. The stomachs of Wistar rat embryos aged 13,21 days were used. The first neuronal bodies and their processes containing synaptophysin-immunoreactivity were observed on embryonic day 13. In contrast, synaptophysin-IR nerve terminals were first observed between mesenchymal cells on embryonic day 14. These results indicate that synaptophysin is expressed in growing neurits and neuronal cell bodies before these neurones have established synaptic connections. The occurrences of mesenchymal cell condensation near synaptophysin-IR neuroblasts on embryonic day 15 reflect an active nerve element-specific mesenchymal cell induction resulting in the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after synaptophysin-IR neuroblasts, on embryonic day 18, suggests that the epithelial differentiation may be closely related to the neuronal maturation as well as other factors. Finally, synaptophysin is functionally important in neuronal development and maturation, together with the establishment of neuroneuronal and neuromuscular contacts and in epithelial differentiation. [source] Comparison of the contractile properties, oxidative capacities and fibre type profiles of the voluntary sphincters of continence in the ratJOURNAL OF ANATOMY, Issue 3 2010Maria Buffini Abstract The external urethral sphincter (EUS) and external anal sphincter (EAS) are the principal voluntary striated muscles that sustain continence of urine and faeces. In light of their common embryological origin, shared tonic sphincteric action and synchronized electrical activity in vivo, it was expected that they would exhibit similar physiological and structural properties. However, the findings of this study using paired observations of both sphincters isolated from the rat show clearly that this is not the case. The anal sphincter is much more fatigable than the urethral sphincter. On completion of a fatigue protocol, the amplitude of the last twitch of the EAS had declined to 42 ± 3% of the first twitch, whereas the last twitch of the EUS was almost identical to that of the first (95 ± 3%). Immunocytochemical detection of myosin heavy-chain isoforms showed that this difference was not due to the presence of more slow-twitch oxidative type 1 fibres in the EUS compared with the EAS (areal densities 4 ± 1% and 5 ± 1%, respectively; P = 0.35). In addition, the fatigue difference was not explained by a greater contribution to force production by fast oxidative type 2A fibres in the urethral sphincter. In fact, the anal sphincter contained a higher areal density of type 2A fibres (56 ± 5% vs. 37 ± 4% in the EUS, P = 0.017). The higher oxidative capacity of the EUS, measured histochemically, explained its fatigue resistance. These results were surprising because the fatigue-resistant urethral muscle exhibited faster single-twitch contraction times compared with the anal sphincter (56 ± 0.87 ms vs. 72.5 ± 1.16 ms, P < 0.001). Neither sphincter expressed the type 2X myosin isoform but the fast-twitch isoform type 2B was found exclusively in the EUS (areal density 16 ± 2%). The type 2B fibres of the EUS were small (diameter 19.5 ± 0.4 ,m) in comparison to typical type 2B fibres of other muscles. As a whole the EUS is a more oxidative than glycolytic muscle. In conclusion, analysis of the twitch mechanics and fatigue of two sphincters showed that the EUS contained more fatigue-resistant muscle fibres compared with the EAS. [source] Pleomorphic xanthoastrocytoma with anaplastic features presenting without GFAP immunoreactivity: Implications for differential diagnosisNEUROPATHOLOGY, Issue 3 2005Ellen Gelpi Pleomorphic xanthoastrocytoma (PXA) is an uncommon, usually low-grade, astrocytic tumor. Characteristic histological features include tumor cell pleomorphism and lipidization of tumor cells. Albeit prognosis in PXA is generally good, cases with histological signs of anaplasia have been observed. In these cases, the differential diagnosis needs to exclude other malignancies, for example, glioblastoma or malignant fibrous histiocytoma. Immunocytochemical detection of GFAP may support exclusion of non-glial neoplasms resembling PXA. However, GFAP expression in PXA may be faint or focal, although complete lack of GFAP has not been described. A 43-year-old woman was operated on for a left occipital parasagital tumor attached to the dura. Histopathology showed a pleomorphic tumor with moderate mitotic activity and necrosis, lack of GFAP immunoreactivity and ultrastructural detection of premelanosome-like structures. These features led to the tentative diagnosis of amelanotic melanoma, and the patient was irradiated. Three years later she had local tumor recurrence and underwent another operation. The recurrent tumor showed similar plain histology as the first specimen. In contrast, anti-GFAP immunoreactivity was now detectable in pleomorphic tumor cells. Anti-GFAP staining of the first biopsy was repeated using monoclonal and polyclonal antibodies in combination with prolonged tissue pretreatment. Focal GFAP staining of tumor cells was now achieved. We conclude that non-standard GFAP staining protocols may enhance sensitivity and thus lead to detection of a low level of GFAP expression in tumor specimens, in which PXA is considered in the differential diagnosis. This may avoid misleading diagnostic considerations that impact on postoperative patient management. [source] A European interlaboratory testing of three well-known procedures for immunocytochemical detection of epithelial cells in bone marrow.CYTOMETRY, Issue 6 2006Results from analysis of normal bone marrow Abstract Background: This investigation intended to study the unspecific background to be expected in normal bone marrow (BM), comparing three well recognized protocols for immunocytochemical detection of disseminated carcinoma cells. The interlaboratory variation in screening and evaluation of stained cells was analyzed and different screening methods were compared. Methods: BM mononuclear cells (BM MNC) from 48 normal BMs were immunostained in parallel by three participating laboratories. The protocols, based on three different anti-cytokeratin antibodies, have all been in common use for detection of disseminated carcinoma cells: the A45-B/B3 protocol (Hamburg), the CK2 protocol (Augsburg) and the AE1AE3 protocol (Oslo). For all protocols, the immunostained cells were visualized by the same alkaline-phosphatase (AP) detection system (APAAP) followed by detection of the cells by manual screening and by two different automated screening systems (ACIS from Chromavision and MDS1 from Applied Imaging). Detected AP-visualized cells were morphologically classified into unambiguous hematopoietic (Uhc) and questionable cells (Qc, potentially interpreted as tumor cells). Results: Seven of 48 BMs (15%) harbored ,1 AP-visualized cell(s) among 1 × 106 BM MNC, both for the A45-B/B3- and for the AE1AE3 protocol, while for CK2 a higher proportion of BMs (21 BMs; 44%) harbored AP-visualized cells (P < 0.01, McNemar's test). The number of Qc was, for all protocols, 1 log lower than the total number of AP-visualized cells. On average, the frequency of Qc was 0.04, 0.08, and 0.02 per 106 BM MNC with A45-B/B3, CK2 and AE1AE3, respectively, and the number of Qc-positive BMs 1, 4, and 1. The MDS1 screening sensitivity was similar to manual screening, while ACIS detected fewer cells (P < 0.001, McNemar's test). Conclusions: All protocols resulted in AP-visualization of occasional hematopoietic cells. However, morphological classification brings the specificity to a satisfactory high level. Approximately 10% of AP-visualized cells were categorized "questionable". The CK2 protocol turned out less specific than the A45-B/B3 and AE1AE3 protocols. © 2006 International Society for Analytical Cytology. [source] Evaluation of apoptosis in cytologic specimensDIAGNOSTIC CYTOPATHOLOGY, Issue 9 2010Viktor Shtilbans Ph.D. Abstract A hallmark of neoplasia is dysregulated apoptosis, programmed cell death. Apoptosis is crucial for normal tissue homeostasis. Dysregulation of apoptotic pathways leads to reduced cytocidal responses to chemotherapeutic drugs or radiation and is a frequent contributor to therapeutic resistance in cancer. The literature pertaining to detection of apoptotic pathway constituents in cytologic specimens is reviewed herein. Virtually all methods for detecting apoptosis, including classic cytomorphologic evaluation, TUNEL assay, immunocytochemistry, and gene sequence analysis, may be applied to cytologic samples as well as tissue. Components of both intrinsic and extrinsic apoptotic pathways have been studied, including many reports examining p53 and bcl-2, as well as studies of caspase inhibitory proteins XIAP and survivin, death receptors and ligands such as Fas, Fas-ligand, and TRAIL. p53 undergoes oncogenic alteration more than any other protein; its immunocytochemical detection almost always connotes loss of its physiologic role as an inducer of apoptosis in response to a damaged genome. Several reports establish cytologic sampling as being as useful as tissue sampling. In one respect cytologic sampling is superior to tissue sampling in particular, by allowing clinicians to repeat sampling of the same tumor before and after administration of therapy; a number of reports use this approach to attempt to predict tumor response by assaying the effect of chemotherapy on the induction of apoptosis. Diagn. Cytopathol. 2010;38:685,697. © 2010 Wiley-Liss, Inc. [source] Immunocytochemistry in liquid-based cervical cytology: Analysis of clinical use following a cross-sectional studyINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Shaira Sahebali Abstract Cytological screening for cervical cancer is hampered by imperfect sensitivity and low inter-observer reproducibility. Human papillomavirus (HPV) testing lacks specificity as a primary screening method. Studies indicate that immunocytochemical detection of alterations caused by HPV in the host cells can optimise screening. Here, the potential of p16INK4a (cyclin-dependent kinase inhibitor p16) and MIB-1 (Ki-67 proliferation marker) as adjunct molecular markers for cervical lesions was investigated in a prospective, cross-sectional study of 500 samples in the framework of opportunistic screening in Flanders, Belgium. A consecutive series of 200 samples and 100 samples from the cytological categories ASC, LSIL and HSIL were investigated. Surepath samples were interpreted according to the Bethesda 2001 reporting system. HPV testing was done with MY09/MY11 consensus PCR. Immunocytochemistry for p16INK4a and MIB-1 was performed with an automated staining protocol. The number of immunoreactive cells/1,000 cervical cells was assessed. There was a higher mean number of p16INK4A and MIB-1 immunoreactive cells/1,000 cells in HSIL (4.06 ± 1.93 and 11.13 ± 2.83, respectively) compared to other cytological categories. Both markers showed a large spread in counts, for all categories. In cases of HSIL without immunoreactive cells for either marker, low cellularity and long-term storage in water were often the cause of false negativity. This study confirms that positive staining for p16INK4a and MIB-1 is highly correlated with presence of high-grade lesions. These markers could be used as adjuncts to increase the sensitivity of cytological screening as well as the specificity of the HPV test. However, clear methodological standards are needed for optimal performance of immunocytochemistry in a clinical setting. © 2005 Wiley-Liss, Inc. [source] Novel targets for valproic acid: up-regulation of melatonin receptors and neurotrophic factors in C6 glioma cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2005Lyda M. Rincón Castro Abstract Valproic acid (VPA) is a potent anti-epileptic and effective mood stabilizer. It is known that VPA enhances central GABAergic activity and activates the mitogen-activated protein kinase,extracellular signal-regulated kinase (MAPK,ERK) pathway. It can also inhibit various isoforms of the enzyme, histone deacetylase (HDAC), which is associated with modulation of gene transcription. Recent in vivo studies indicate a neuroprotective role for VPA, which has been found to up-regulate the expression of brain-derived neurotrophic factor (BDNF) in the rat brain. Given the interaction between the pineal hormone, melatonin, and GABAergic systems in the central nervous system, the effects of VPA on the expression of the mammalian melatonin receptor subtypes, MT1 and MT2, were examined in rat C6 glioma cells. The effects of VPA on the expression of glial cell line-derived neurotrophic factor (GDNF) and BDNF were also examined. RT-PCR studies revealed a significant induction of melatonin MT1 receptor mRNA in C6 cells following treatment with 3 or 5 mm VPA for 24 h or 5 mm VPA for 48 h. Western analysis and immunocytochemical detection confirmed that the VPA-induced increase in MT1 mRNA results in up-regulation of MT1 protein expression. Blockade of the MAPK,ERK pathway by PD98059 enhanced the effect of VPA on MT1 expression, suggesting a negative role for this pathway in MT1 receptor regulation. In addition, significant increases in BDNF, GDNF and HDAC mRNA expression were observed after treatment with VPA for 24 or 48 h. Taken together, the present findings suggest that the neuroprotective properties of VPA involve modulation of neurotrophic factors and receptors for melatonin, which is also thought to play a role in neuroprotection. Moreover, the foregoing suggests that combinations of VPA and melatonin could provide novel therapeutic strategies in neurological and psychiatric disorders. [source] Plasma Vasopressin Concentrations and Fos Protein Expression in the Supraoptic Nucleus Following Osmotic Stimulation or Hypovolaemia in the Ovariectomized Rat: Effect of Oestradiol ReplacementJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2004D. E. Hartley Abstract The set points for vasopressin release in response to increasing plasma osmolality and hypovolaemia alter with reproductive status. Here, we studied stimulated vasopressin release following ovariectomy and oestrogen replacement, neuronal activity being measured in terms of immediate early gene expression. Observations were carried out on three groups of female Sprague-Dawley rats. The first group were ovariectomized. The second group were given a subcutaneous oestrogen implant (20 µg/ml oestradiol-17,) at the time of ovariectomy. The final group were left intact and observations performed at oestrus. Two weeks after ovariectomy, vascular cannulae were implanted under anaesthesia and at least 48 h allowed for recovery before hormone release was stimulated by infusion of 1.5 m NaCl for 90 min, or hypovolaemia induced by the removal of 10 mg/kg body weight taken in 1-ml aliquots. Blood pressure was monitored, and blood samples were taken for determination of packed cell volume and plasma vasopressin and osmolality. After a minimum of 48 h, the challenge was repeated, the rats anaesthetized, and perfused with 4% paraformaldehyde. Brain sections were processed for immunocytochemical detection of Fos protein. Vasopressin release in response to both stimuli was reduced in ovariectomized compared to intact rats and the response could be substantially restored by oestradiol replacement. The number of Fos positive cells in the supraoptic nucleus of oestrogen-replaced rats was significantly higher than in the ovariectomized group and not statistically different from the intact group. [source] Perioperative detection of disseminated tumour cells is an independent prognostic factor in patients with colorectal cancer ,BRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 7 2003B. Bosch Background: The objective of the present investigation was to assess the prognostic significance of disseminated tumour cells in peritoneal lavage, and peripheral and mesenteric venous blood in patients undergoing curative resection of colorectal cancer. Methods: The prognostic impact of perioperative cytological and immunocytochemical detection of disseminated colorectal cancer cells was evaluated prospectively. Peritoneal lavage fluid, and peripheral and mesenteric venous blood from 53 consecutive patients undergoing curative surgery for colorectal cancer were analysed. The dichotomous results (positive versus negative) from the cytological and immunocytochemical analysis were used as a predictor along with other co-variates in proportional hazard regression models of disease-free and overall survival. Results: Disseminated colorectal cancer cells were found in 13 of 53 patients (25 per cent) using cytology (CYT) and/or immunocytochemistry (ICC). The median follow-up at the time of the analysis was 37 months. In multivariate proportional hazard regression models CYT/ICC status was a significant predictor for disease-free (P = 0·002) and overall (P = 0·006) survival. Conclusion: Disseminated tumour cells detected by CYT and ICC represent an independent prognostic factor in patients undergoing surgery for colorectal cancer and may identify patients at high risk of recurrence. Copyright © 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] Frequency and prognostic relevance of disseminated tumor cells in bone marrow of patients with metastatic renal cell carcinomaCANCER, Issue 7 2006Alexander Buchner M.D. Abstract BACKGROUND The prognostic relevance of disseminated cytokeratin-positive (CK+) tumor cells in the bone marrow of patients with different types of carcinoma has been demonstrated in several studies. In this prospective study, the frequency and prognostic value of CK+ tumor cells was investigated in the bone marrow of 55 consecutive patients with metastatic renal cell carcinoma (M1 RCC) in comparison with 256 M0 RCC patients from a previous study. METHODS Aspiration of bone marrow from the anterior iliac crest was performed immediately before tumor resection in RCC patients. Cytospins were made and stained by immunocytochemistry using the APAAP (alkaline phosphatase-antialkaline phosphatase) protocol and monoclonal antibodies CK2 and A45-B/B3. Twenty-seven patients with no evidence of any malignant disease served as a control group. RESULTS CK+ tumor cells were detected in 42% (23 of 55 patients) of the M1 patients and 25% (63 of 256 patients) of the M0 patients (P <.01). No CK+ cells (0 of 27 patients) were detected in the control group. In the M1 group, CK, patients demonstrated a trend toward a better outcome compared with CK+ patients (log-rank test, P = .19). This difference was significant when applying a higher threshold (0,2 CK+ cells vs. , 3 CK+ cells; P <.05). On multivariate analysis, the detection of , 3 CK+ cells in the bone marrow was found to be an independent prognostic factor (P <.001). CONCLUSIONS The results of the current study indicate that disseminated CK+ cells play a role in the biology of tumor spread of RCC, and that their immunocytochemical detection can be useful in assessing the prognosis of patients with M1 disease. Cancer 2006. © 2006 American Cancer Society. [source] | ||||||||||||||||||||||