1,721,948 research outputs found
Induction of Cell Death by Saponin and Antigen Delivery
No full textPurpose. Saponin is the major component in the formation of immune stimulating complex ( ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear . The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in EL4 mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM. Methods. EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/ propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin- protein complexes or saponin- treated cells and analyzed by flow cytometry. Results. Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA- binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad- range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin- induced dead cells. Conclusions. Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in EL4 cells . These events are critical for antigen processing and presentation
Glucose-Modulated Transgene Expression Via Recombinant Adeno-Associated Virus
No full textPurpose. The objective of this study was to examine glucose- modulated reporter gene expression via recombinant adeno- associated viral vectors both in vitro and in vivo. Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10(-)7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin- induced diabetic animals. Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV -transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV- treated diabetic C57BL/6J mice
The Ultrastructure of Tomatine Adjuvant
No full textThe tomatine adjuvant, consisting of tomatine, n-octyl-beta- D- glucopyranoside, phosphatidylethanolamine, cholesterol, and ovalbumin, has recently been shown to potentiate the immunogenicity of protein antigen and elicit cytotoxic T- lymphocyte responses to immunized animals. The physicochemical properties of tomatine adjuvant have not been characterized. The aim of this study was to examine the microstructure of this complex formulation, as directly related to its physicochemical properties. To elucidate the micromorphology of this system, the tomatine adjuvant was separated by isopycnic ultracentrifugation, followed by freeze fracturing and examination by transmission and scanning electron microscopy. The adjuvant mixture was shown to be composed of several micro - and nano-structures. The major fraction obtained from isopycnic separation was shown to consist of flaky needle-like microcrystals, approximately 80-160 nm in width-and 2-4 mum in length. The tomatine crystals alone in 0.9% NaCl, on the other hand, were shown to be elongated hollow tubular crystals of hundreds of nanometers up to a few microns in length, along which n- octyl-beta-glucopyranoside was speculated to serve as a seeding microtemplate for gel crystallization of protein complexes. Indented marks within the gel phase were observed in the freeze fractured replicas of the adjuvant, suggesting that protein complexes may have been crystallized or precipitated within the gels. Several other forms of micro - and nano-structures were also observed, showing multiple-dispersion features with gel characteristics. The presence of gel crystalline and multiple-dispersed phases is postulated to contribute to the sustained immunopotentiation effect of tomatine adjuvant. (C) 2002 Elsevier Science Ltd. All rights reserved
The Apoptotic and Necrotic Effects of Tomatine Adjuvant
No full textTomatine adjuvant, consisting of tomatine, n-octyl-beta-D- glucopyranoside (OGP), phosphatidylethanolamine and cholesterol is unique in that when combined with soluble protein antigen it elicits a cytotoxic T lymphocyte (CTL) response in immunized animals. The mechanisms underlying this property are unknown. In an attempt to understand how tomatine activates cellular immunity, we examined its potential to induce apoptosis. Thus in the present study, cell death of EL4 thymoma cells induced by whole adjuvant and the surface-active components in the formulation was examined. Cytotoxicity was monitored using the MTT [3-(4,5 - dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and lactate dehydrogenase release assays, apoptosis and necrosis were quantified by flow cytometry using Annexin V and propidium iodide staining, and morphology was examined by Hoechst 33342 staining. Flow cytomietric analysis demonstrated the appearance of the sub-G I phase in cells treated with these agents and Annexin V/PI staining showed that all three agents induced both apoptosis and necrosis in EL4 cells in a concentration- dependent manner. Tomatine was effective at much lower concentrations than OGP, suggesting that the majority of the effect of whole adjuvant could be attributed to this component. Microscopic examination of EL4 cells after treatment with these agents revealed morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Pretreatment with zVAD- fmk did not block cell death induced by these agents, showing that tomatine adjuvant-induced EL4 cell apoptosis is caspase-independent. (C) 2003 Elsevier Ltd. All rights reserved
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Obstructive colitis proximal to partially obstructive colonic carcinoma: a case report and review of literature
No full text- …
