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Labeling Method (labeling + method)
Selected AbstractsHuman immunodeficiency virus gag and pol-specific CD8 T cells in perinatal HIV infectionCYTOMETRY, Issue 5 2001Thomas W. McCloskey Abstract Background: Binding of fluorochrome-conjugated MHC class I tetramers is a powerful means to detect antigen-specific CD8 T lymphocytes. In human immunodeficiency virus (HIV) infection, cellular immune response is essential in curtailing HIV disease progression but gaps persist in our understanding of HIV-specific cells during the disease course. In this study, we evaluated tetramer binding HIV-specific CD8 T cells in HIV-infected children. Methods: Fluorescently labeled tetramers for HIV gag and pol were utilized to quantify antigen-specific cells by flow cytometry using a whole blood labeling method in a cohort of 19 HLA-A2+ HIV- infected children (age range 1 month to 17 years). Results: Fourteen children had detectable gag (median 0.4%) and pol (median 0.1%) binding CD8 T cells, three children had gag binding cells only, and two had neither. Numbers of gag and pol binding cells correlated with each other and each correlated independently with total CD8 T cells and total CD4 T cells. Conclusions: HIV gag and pol-specific CD8 T cells are maintained during the chronic phase of HIV infection in children and CD4 lymphocytes appear to be important for sustaining their levels. Cytometry (Comm. Clin. Cytometry) 46:265,270, 2001. © 2001 Wiley-Liss, Inc. [source] Demonstration of long-range GABAergic connections distributed throughout the mouse neocortexEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Ryohei Tomioka Abstract ,-Aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network. [source] An improved direct labeling method for the max,flow min,cut computation in large hypergraphs and applicationsINTERNATIONAL TRANSACTIONS IN OPERATIONAL RESEARCH, Issue 1 2003Joachim Pistorius Algorithms described so far to solve the maximum flow problem on hypergraphs first necessitate the transformation of these hypergraphs into directed graphs. The resulting maximum flow problem is then solved by standard algorithms. This paper describes a new method that solves the maximum flow problem directly on hypergraphs, leading to both reduced run time and lower memory requirements. We compare our approach with a state,of,the,art algorithm that uses a transformation of the hypergraph into a directed graph and an augmenting path algorithm to compute the maximum flow on this directed graph: the run,time complexity as well as the memory space complexity are reduced by a constant factor. Experimental results on large hypergraphs from VLSI applications show that the run time is reduced, on average, by a factor approximately 2, while memory occupation is reduced, on average, by a factor of 10. This improvement is particularly interesting for very large instances, to be solved in practical applications. [source] Intraperitoneal injection of d- galactosamine provides a potent cell proliferation stimulus for the detection of initiation activities of chemicals in rat liverJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2005Yoshiji Asaoka Abstract In an in vivo 5-week initiation assay model, chemical hepatectomy by hepatotoxicant administration was utilized as a cell proliferation stimulus as an alternative to the two-thirds partial hepatectomy. The study investigated the effect of an intraperitoneal (i.p.) injection of d- galactosamine (d -gal) for this purpose in a medium-term liver bioassay, with a further focus on cell proliferation kinetics and cytochrome P450 (CYP) expression. In experiment I, cell proliferation in rat liver after a single administration of d -gal (700 mg kg,1, i.p.) was analysed by the bromodeoxyuridine (BrdU) labeling method, and CYP isozymes were quantified by immunoblotting. In experiment II, the induction of glutathione S-transferase placental form (GST-P) positive foci by 1,2-dimethylhydrazine (DMH) was evaluated in a modified in vivo 5-week initiation assay model. At 84 hours after single administration of d -gal (i.p.) the BrdU index was markedly elevated (27.5% ± 9.5%). Although CYP 2E1 and 1A2 apoprotein contents decreased transiently to less than 20% of the control level, subsequently they recovered to 60% and 40% of the control level, respectively, at 84 hours. Induction of GST-P positive foci in the group given DMH at 84 hours after a single administration of d -gal was significantly greater than in the control group, correlating with the kinetics of cell proliferation. In conclusion, the sensitivity of the present initiation assay using d -gal i.p. is high, so that d -gal i.p. can be considered an effective cell proliferation stimulus. Copyright © 2005 John Wiley & Sons, Ltd. [source] A Novel Tetracycline Labeling Schedule for Longitudinal Evaluation of the Short-Term Effects of Anabolic Therapy With a Single Iliac Crest Bone Biopsy: Early Actions of Teriparatide,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2006Robert Lindsay MD Abstract We describe a quadruple tetracycline labeling method that allows longitudinal assessment of short-term changes in bone formation in a single biopsy. We show that 1 month of hPTH(1-34) treatment extends the bone-forming surface, increases mineral apposition rate, and initiates modeling-based formation. Introduction: Iliac crest biopsy, with histomorphometric evaluation, provides important information about cellular activity in bone. However, to obtain longitudinal information, repeat biopsies must be performed. In this study, we show the capability to obtain short-term longitudinal information on bone formation in a single biopsy using a novel, quadruple labeling technique. Materials and Methods: Two tetracycline labels were administered using a standard 3 days on, 12 days off, 3 days on format. Four weeks later, the tetracycline labeling was repeated using the same schedule but with a different tetracycline that can be distinguished from the first by its color under fluorescent light. Iliac crest biopsies were performed 1 week later and prepared undecalcified for histomorphometry. Indices of bone formation 1 month apart were measured and calculated using the two sets of labels. We used this method to investigate the early effects of teriparatide [hPTH(1-34)] treatment on bone formation. The results were compared with those from a group of control subjects who were quadruple-labeled, but did not receive hPTH(1-34). Results: Treatment with hPTH(1-34) dramatically stimulated bone formation on cancellous and endocortical surfaces. This was achieved by both an increase in the linear rate of matrix apposition and extension of the bone-forming surface. New bone was deposited on previously quiescent surfaces (i.e., modeling-based formation), but a proportion of this could occur by encroachment from adjacent resorption cavities. Conclusions: A single transiliac crest bone biopsy, after sequential administration of two sets of tetracycline labels is a useful approach to study the short-term effects of anabolic agents on human bone. One month of hPTH(1-34) treatment extends the bone-forming surface, increases mineral apposition rate, and initiates modeling-based formation. [source] Ultrastructural identification of peripheral myelin proteins by a pre-embedding immunogold labeling methodJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2003Marie-Hélène Canron Abstract Ultrastructural immunolabeling of peripheral nervous system components is an important tool to study the relation between structure and function. Owing to the scarcity of certain antigens and the dense structure of the peripheral nerve, a pre-embedding technique is likely appropriate. After several investigations on procedures for pre-embedding immunolabeling, we propose a method that offers a good compromise between detection of antigenic sites and preservation of morphology at the ultrastructural level, and that is easy to use and suitable for investigations on peripheral nerve biopsies from humans. Pre-fixation by immersion in paraformaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure. Then, ultrasmall gold particles with silver enhancement are advised. Antibodies against myelin protein zero and myelin basic protein were chosen for demonstration. The same technique was applied to localize a 35 kDa myelin protein. [source] Prognostic Significance of p53/bcl-2 Co-expression in Patients With Laryngeal Squamous Cell CarcinomaTHE LARYNGOSCOPE, Issue 8 2000Martin C. Jäckel MD Abstract Objective The p53, bcl-2, and bax genes are known to be involved in control of cell cycle progression and regulation of apoptotic cell death. Although they are frequently altered in laryngeal squamous cell carcinoma, their clinical relevance is not yet fully understood. In the present study, individual and combined expressions of these genes were related with patient survival as well as with proliferative and apoptotic activity. Design Retrospective study. Methods Paraffin-embedded tissue sections of 88 laryngeal squamous cell carcinomas that were diagnosed and treated between 1986 and 1996 were investigated for p53, bcl-2, and bax protein expression by immunohistochemistry. Apoptotic cells were visualized using the nick end labeling method. To assess proliferative activity of tumors, mitotic indices were determined. Results Age of patients, advanced disease (stages III and IV), high mitotic activity, positive bcl-2 expression, high level of p53 expression, and p53/bcl-2 co-expression were significantly associated with shortened overall survival in univariate analysis. In multivariate analysis, only age and p53/bcl-2 co-expression had independent prognostic value. Other combinations of genes, i.e., bcl-2-to-bax and p53-to-bax ratios, were not associated with patient outcome. A significant positive correlation was found between apoptotic and mitotic activity. However, protein levels of p53, bcl-2, and bax were unrelated to proliferation and apoptosis of tumor cells. Conclusions The co-expression of p53/bcl-2 was an independent predictor of patient outcome and had a prognostic value superior to both parameters considered separately. The rate of apoptosis mainly counterbalanced proliferative activity but appeared not to be significantly influenced by p53, bcl-2, and bax. [source] In Vivo Distribution of Liposome-Encapsulated Hemoglobin Determined by Positron Emission TomographyARTIFICIAL ORGANS, Issue 2 2009Takeo Urakami Abstract Positron emission tomography (PET) is a noninvasive imaging technology that enables the determination of biodistribution of positron emitter-labeled compounds. Lipidic nanoparticles are useful for drug delivery system (DDS), including the artificial oxygen carriers. However, there has been no appropriate method to label preformulated DDS drugs by positron emitters. We have developed a rapid and efficient labeling method for lipid nanoparticles and applied it to determine the movement of liposome-encapsulated hemoglobin (LEH). Distribution of LEH in the rat brain under ischemia was examined by a small animal PET with an enhanced resolution. While the blood flow was almost absent in the ischemic region observed by [15O]H2O imaging, distribution of 18F-labeled LEH in the region was gradually increased during 60-min dynamic PET scanning. The results suggest that LEH deliver oxygen even into the ischemic brain from the periphery toward the core of ischemia. The real-time observation of flow pattern, deposition, and excretion of LEH in the ischemic rodent brain was possible by the new methods of positron emitter labeling and PET system with a high resolution. [source] Modulation of tamoxifen sensitivity by antisense Bcl-2 and trastuzumab in breast carcinoma cellsCANCER, Issue 10 2005Ph.D., Ryungsa Kim M.D. Abstract BACKGROUND Because the overexpression of HER-2 and Bcl-2 is associated with resistance to tamoxifen (TAM), the authors examined the effect of antisense (AS) Bcl-2 on sensitivity to TAM compared with the effect of trastuzumab on sensitivity to TAM in breast carcinoma cell lines. METHODS Drug sensitivity was assessed in vitro using a [3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay with the breast carcinoma cell lines ZR-75-1, MDA-MB-453, and BT-474. AS Bcl-2 18-mer phosphorothioate oligonucleotide was applied. Apoptotic cell death was assessed with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling method, and gene expression was evaluated with Western blot analysis. RESULTS The expression of Bcl-2 was identified in ZR-75-1 and BT-474 cells and, to a lesser extent, in MDA-MB-453 cells. Overexpression of HER-2 was identified in BT-474 cells, and moderate expression was identified in MDA-MB-453 and ZR-75-1 cells. Combination treatment with trastuzumab or AS Bcl-2 enhanced TAM sensitivity in ZR-75-1 cells, which showed 50% inhibitory concentration (IC50) values of 0.9 ,M (7.2-fold increase) and 0.5 ,M (13.0-fold), respectively. Combination treatment with trastuzumab or AS Bcl-2 slightly enhanced TAM sensitivity of BT-474 cells, with IC50 values of 3.0 ,M (1.3-fold) and 1.5 ,M (2.6-fold), respectively. The sensitivity of MDA-MB-453 cells to TAM was not enhanced by combination with trastuzumab or AS Bcl-2. Modulation of TAM sensitivity by AS Bcl-2 was superior to modulation by trastuzumab in HER-2-expressing and Bcl-2 -expressing breast carcinoma cells. Enhanced sensitivity in combination with AS Bcl-2 was associated with down-regulation of Bcl-2 and pAkt, which was correlated with the induction of Bax and caspase-3, leading to apoptosis. CONCLUSIONS AS Bcl-2 appeared to be superior to trastuzumab with respect to regulating the signal-transduction pathways involved in breast carcinoma cells. Cancer 2005. © 2005 American Cancer Society. [source] | |||||||||||||