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Cytometric Evaluation (cytometric + evaluation)
Selected AbstractsFlow cytometric evaluation of CD38 expression assists in distinguishing follicular hyperplasia from follicular lymphoma,CYTOMETRY, Issue 5 2009Kristin Mantei Abstract The distinction of follicular lymphoma (FL) from reactive follicular hyperplasia (FH) can be a diagnostic challenge in flow cytometry. In this study, the median fluorescent intensity (MFI) of CD38 as assessed by flow cytometry on B and T cell subpopulations in 102 lymph nodes specimens with histopathologically confirmed FL was compared with 55 cases of FH. The MFI of CD38 was highly significantly reduced in the neoplastic B cells in FL when compared with the reactive germinal center B cells in FH (P < 1.0E-16). The MFI of CD38 did not differ between the non-neoplastic B-cells in FL and nongerminal center B-cells in FH (P = 0.14) or between T-cells and non-neoplastic B-cells in FL (P = 0.63). A marginal increase in the MFI of CD38 was seen for T cells in FL compared with FH (P = 0.04). An increased difference in the MFI of CD38 was identified for T-cells compared with nongerminal center B-cells in FH (P = 0.005). No difference in CD38 expression was seen between Grades 1, 2, or 3 FL. The study also confirmed increased expression of CD10 (P < 1.0E-9), decreased CD19 (P < 1.0E-22), and CD20 (P < 1.0E-16) in FL in comparison with FH, as has been previously reported. This study identified decreased CD38 as a common finding in FL in comparison with FH and provides an additional tool to help differentiate FL from FH by flow cytometry. © 2009 Clinical Cytometry Society [source] No increases in biomarkers of genetic damage or pathological changes in heart and brain tissues in male rats administered methylphenidate hydrochloride (Ritalin) for 28 days,,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2010Kristine L. Witt Abstract Following a 2005 report of chromosomal damage in children with attention deficit/hyperactivity disorder (ADHD) who were treated with the commonly prescribed medication methylphenidate (MPH), numerous studies have been conducted to clarify the risk for MPH-induced genetic damage. Although most of these studies reported no changes in genetic damage endpoints associated with exposure to MPH, one recent study (Andreazza et al. [2007]: Prog Neuropsychopharmacol Biol Psychiatry 31:1282,1288) reported an increase in DNA damage detected by the Comet assay in blood and brain cells of Wistar rats treated by intraperitoneal injection with 1, 2, or 10 mg/kg MPH; no increases in micronucleated lymphocyte frequencies were observed in these rats. To clarify these findings, we treated adult male Wistar Han rats with 0, 2, 10, or 25 mg/kg MPH by gavage once daily for 28 consecutive days and measured micronucleated reticulocyte (MN-RET) frequencies in blood, and DNA damage in blood, brain, and liver cells 4 hr after final dosing. Flow cytometric evaluation of blood revealed no significant increases in MN-RET. Comet assay evaluations of blood leukocytes and cells of the liver, as well as of the striatum, hippocampus, and frontal cortex of the brain showed no increases in DNA damage in MPH-treated rats in any of the three treatment groups. Thus, the previously reported observations of DNA damage in blood and brain tissue of rats exposed to MPH for 28 days were not confirmed in this study. Additionally, no histopathological changes in brain or heart, or elevated serum biomarkers of cardiac injury were observed in these MPH-exposed rats. Environ. Mol. Mutagen. 2010. Published 2009 Wiley-Liss, Inc. [source] Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transferEQUINE VETERINARY JOURNAL, Issue 1 2008B. A. BALL Summary Reasons for performing study: Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objectives: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647 -pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively. Results: Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647 -pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed. Conclusions: The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7,10 embryos, there was no evidence of expression of EGFP in these embryos. Potential relevance: Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids. [source] Evaluation of the impact of highly active antiretroviral therapy on immune recovery in antiretroviral naive patientsHIV MEDICINE, Issue 1 2004L Al-Harthi Objectives To examine the extent of immune reconstitution in treatment-naive patients with CD4 T-cell counts <500 cells/,L following 48 weeks of highly active antiretroviral therapy (HAART). Methods Thirteen antiretroviral naive patients were evaluated longitudinally for 48 weeks on HAART utilizing immune functional and lymphocyte phenotyping assays, including lymphocyte proliferation assay, flow cytometric evaluation of cell surface markers, and delayed type hypersensitivity skin tests. Virologic responses were monitored using commercially available viral load assays and gag/pol mRNA quantification using simultaneous immunophenotyping/UltraSensitive fluorescence in situ hybridization (ViroTect In Cell HIV-1 Detection Kit; Invirion, Frankfort, MI). Thymic function was evaluated for a subset of four patients using real-time polymerase chain reaction (PCR) for T-cell receptor excision circle (TREC) quantification and thymic scans using computerized axial tomography (CT) of the thymus. Results HAART initiation resulted in a significant decline in plasma viremia and percentage of infected peripheral blood cells, and a rise in CD4 T cells from a baseline median of 207 cells/,L to a week-48 median of 617 cells/,L. The rise was predominately in CD4 memory cells. Naive T cells also increased in number, but at a slower rate. Activated (HLA-DR CD38) CD4 and CD8 T cells were elevated at baseline (24 and 62%, respectively) and declined by week 48 (17 and 36%, respectively) but did not reach normal levels. The number of Fas CD4 T cells increased from a baseline median of 169 to 381 cells/,L at week 48. Both soluble interleukin (IL)-2 and tumour necrosis factor (TNF) II receptors declined by week 48. HIV p24 lymphocyte proliferation assay responses were transiently detected in three patients. TREC values increased from a median 6400 copies/,g at baseline to a week-48 median value of 26 697 copies/,g. Conclusion Immune functional reconstitution was not achieved in these HAART naive patients. [source] Antifungal activity of Thymus oils and their major compoundsJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 1 2004C Pina-Vaz ABSTRACT The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. Essential oils have been used empirically. The essential oils of Thymus (Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina) have often been used in folk medicine. The aim of the present study was to evaluate objectively the antifungal activity of Thymus oils according to classical bacteriological methodologies , determination of the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) , as well as flow cytometric evaluation. The effect of essential oils upon germ tube formation, an important virulence factor, was also studied. The mechanism of action was studied by flow cytometry, after staining with propidium iodide. The chemical composition of the essential oils was investigated by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The antifungal activity of the major components (carvacrol, thymol, p -cymene and 1,8-cineole) and also possible interactions between them were also investigated. The essential oils of T. vulgaris and T. zygis showed similar antifungal activity, which was greater than T. mastichina. MIC and MLC values were similar for all the compounds tested. At MIC values of the essential oils, propidium iodide rapidly penetrated the majority of the yeast cells, indicating that the fungicidal effect resulted primarily from an extensive lesion of the cell membrane. Concentrations below the MIC values significantly inhibited germ tube formation. This study describes the potent antifungal activity of the essential oils of Thymus on Candida spp., warranting future therapeutical trials on mucocutaneous candidosis. [source] Posttransplant Bronchiolitis Obliterans Syndrome Is Associated with Bronchial Epithelial to Mesenchymal TransitionAMERICAN JOURNAL OF TRANSPLANTATION, Issue 4 2009S. Hodge Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells. Large airway brushings were obtained at 33 bronchoscopies in 16 BOS-free and 6 BOS grade 1,3 patients at 2,120 months posttransplant. Flow cytometry was used to assess expression of the mesenchymal markers ,SMA, S100A4 and ED-A FN and HLA-DR. TGF ,1 and HGF were measured in Bronchoalveolar lavage (BAL). Expression of all three mesenchymal markers was increased in BOS, as was HLA-DR. BAL HGF, but not TGF ,1 was increased in BOS. Longitudinal investigation of one patient revealed a 100% increase in EMT markers concurrent with a 6-fold increase in BAL TGF ,1 and the diagnosis of BOS at 17 months posttransplant. Flow cytometric evaluation of bronchial epithelium may provide a novel and rapid means to assess lung allografts at risk of BOS. [source] Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosomeANDROLOGIA, Issue 1 2010N. Falzone Summary Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false-positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) were compared for their ability to stain nonviable cells post-fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7-AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7-AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore-induced acrosome reaction. [source] | |||||||||||||