The Effects of Malignant Transformation on Susceptibility of Human Urothelial Cells to CD40-Mediated Apoptosis

Background: The tumor necrosis factor (TNF) superfamily of ligands and receptors mediates immune cell survival. Some members possess a death domain, a protein motif that functions to transmit apoptotic signals, whereas others, such as CD40, do not. CD40 is expressed by both normal and malignant epithelial cells. To investigate the functional significance of this expression, we studied the effects of ligation of CD40, Fas, and TNF receptors (TNFRs) on the proliferation and survival of normal and malignant human urothelial cells and urothelial cells with disabled p53 function. Methods: Normal and malignant human urothelial cells were cultured with soluble TNF family agonists (CD40 ligand [CD40L], TNF-α, anti-Fas antibody, or cocultured with mouse fibroblasts stably transfected with plasmids that caused the cells to constitutively express CD40L or CD32; cell proliferation was estimated by an [3H]thymidine incorporation assay, and apoptosis was determined by Annexin V staining and by a DNA fragmentation assay. Messenger RNA levels for CD40 and potential downstream effector molecules were quantified by polymerase chain reaction-based and ribonuclease protection assays, respectively, and nuclear factor (NF) κB nuclear translocation was detected by immunofluorescence. All statistical tests were two-sided. Results: Soluble trimeric CD40L inhibited the growth of normal and malignant urothelial cells but did not induce apoptosis. Cell surface-presented CD40L induced massive apoptosis in CD40-positive transitional cell carcinoma cells but not in normal urothelial cells. Normal cells underwent CD40L-mediated apoptosis only in the presence of other TNFR agonists. An agonistic anti-CD40 antibody presented on the surface of CD32-transfected fibroblasts also induced apoptosis in transitional cell carcinoma cells and in normal urothelial cells. Apoptotic responses of tumor (but not normal) cells to soluble agonists were enhanced by blocking protein synthesis. Karyotypically normal urothelial cells with disabled p53 function underwent apoptosis during coculture with CD40L-expressing fibroblasts alone but were not additionally sensitive to additional TNFR agonists. Conclusions: Susceptibility to CD40 ligation-induced apoptosis may be a novel mechanism for eliminating neoplastically transformed urothelial cells. Loss of CD40 expression may be an important adaptive mechanism for transitional cell carcinoma development and progressio

The influence of chitosan flake deacetylation degree on orthophosphate sorption efficiency from aqueous solutions

The article presents the effectiveness of orthophosphate sorption from aqueous solutions depending on the deacetylation degree of chitosan flakes. The first stage of the research was to determine the pH value at which the sorption process was the most effective (from the pH range 2–11). In the second stage, research was carried out to determine the maximum sorption capacities of chitosan with deacetylation degrees of 75%, 85% and 90% in relation to PO43-. The highest effectiveness of orthophosphate removal on chitosan, regardless of its deacetylation degree, was obtained at pH 4. At pH 2 and 3, the chitosan flakes dissolved. This study showed that the sorption effectiveness of phosphorus compounds depends on the deacetylation degree of chitosan. Along with the increase in deacetylation degree, the sorption capacity of chitosan also increases in relation to orthophosphates. It is related to the higher number of amino groups in the structure of chitosan, which are responsible for the sorption of pollutants in the form of anions. The maximum sorption capacity of chitosan-DD = 75% in relation to biogen was 5.13 mg/g, chitosan-DD = 85% was 5.65 mg/g, and chitosan-DD = 90% was 5.91 mg/g. After 60 minutes, the desorption process had begun and was most likely caused by an increase in the pH of the solution. Due to chitosan's ability to neutralise the sample and the associated risk of desorption, the time of sorbent contact with sewage cannot be longer than 60 minutes

The influence of chitin amination on the effectiveness of rb5 and ry84 dye sorption

This article presents the influence of chitin amination on the effectiveness of RB5 and RY84 dye sorption. For chitin and chitin modified by amination, the optimal pH of sorption and the maximum sorption capacity were determined in relation to two reactive dyes: Reactive Black 5 (RB5) and Reactive Yellow 84 (RY84), differing in the active group and molecular weight. Three sorption models were used to describe the experimental data: Langmuir, Langmuir 2 and Freundlich. The highest sorption capacity was obtained for aminated chitin for both tested dyes: 386.53 mg/g for RB5 and 261.56 mg/g for RY84. In the case of sorption on unmodified chitin, the sorption capacities were lower: up to 235.65 mg/g.d.m. for RB5 and 208.88 mg/g.d.m. for RY84. The modification of chitin by amination has a beneficial effect on the amount of dye adsorbed in the process. The adsorptive capacity increased by 1.6-times in the case of RB5 and 1.25-times in case of RY84

The influence of the deacetylation degree of chitosan in the form of flakes on the effectiveness of nitrates v sorption from aqueous solutions

The influence of the degree of deacetylation of chitosan from the range of DD = 75–90% on the effectiveness of sorption of nitrates from aqueous solutions was investigated. The scope of the research included: determining the effect of pH on the effectiveness of N-NO3 binding on chitosan sorbents and determining the sorption capacity of chitosan sorbents with different degrees of deacetylation after 5, 15, 30 and 60 minutes. The effectiveness of sorption of nitrates on chitosan sorbents increased in the series DD=75% < DD=85% < DD=90%. Regardless of the degree of deacetylation, the sorption effectiveness of nitrates on chitosan was the highest at pH 4. The amount of nitrate-related sorbents was the highest after 30 min of sorption. A process time which was too long resulted in desorption of nitrates. The maximum sorption capacity for chitosan with the degree of deacetylation DD = 75, 85 and 90% was 0.59 mg N-NO3/g, 0.60 mg N-NO3/g and 0.87 mg N-NO3/g, respectively

The effects of malignant transformation on susceptibility of human urothelial cells to CD40-mediated apoptosis

Background: The tumor necrosis factor (TNF) superfamily of ligands and receptors mediates immune cell survival. Some members possess a death domain, a protein motif that functions to transmit apoptotic signals, whereas others, such as CD40, do not. CD40 is expressed by both normal and malignant epithelial cells. To investigate the functional significance of this expression, we studied the effects of ligation of CD40, Fas, and TNF receptors (TNFRs) on the proliferation and survival of normal and malignant human urothelial cells and urothelial cells with disabled p53 function. Methods: Normal and malignant human urothelial cells were cultured with soluble TNF family agonists (CD40 ligand [CD40L], TNF-{alpha}, anti-Fas antibody, or cocultured with mouse fibroblasts stably transfected with plasmids that caused the cells to constitutively express CD40L or CD32; cell proliferation was estimated by an [3H]thymidine incorporation assay, and apoptosis was determined by Annexin V staining and by a DNA fragmentation assay. Messenger RNA levels for CD40 and potential downstream effector molecules were quantified by polymerase chain reaction-based and ribonuclease protection assays, respectively, and nuclear factor (NF) {kappa}B nuclear translocation was detected by immunofluorescence. All statistical tests were two-sided. Results: Soluble trimeric CD40L inhibited the growth of normal and malignant urothelial cells but did not induce apoptosis. Cell surface-presented CD40L induced massive apoptosis in CD40-positive transitional cell carcinoma cells but not in normal urothelial cells. Normal cells underwent CD40L-mediated apoptosis only in the presence of other TNFR agonists. An agonistic anti-CD40 antibody presented on the surface of CD32-transfected fibroblasts also induced apoptosis in transitional cell carcinoma cells and in normal urothelial cells. Apoptotic responses of tumor (but not normal) cells to soluble agonists were enhanced by blocking protein synthesis. Karyotypically normal urothelial cells with disabled p53 function underwent apoptosis during coculture with CD40L-expressing fibroblasts alone but were not additionally sensitive to additional TNFR agonists. Conclusions: Susceptibility to CD40 ligation-induced apoptosis may be a novel mechanism for eliminating neoplastically transformed urothelial cells. Loss of CD40 expression may be an important adaptive mechanism for transitional cell carcinoma development and progression