Serum albumin level in burns patients

The aim of this retrospective study was to examine whether serum albumin levels offer a good marker of nutritional status in patients with burns. Serum albumin levels have been used to evaluate nutritional status in burns patients, even though these levels are affected by various factors and are not specific to malnutrition. To clarify whether provision of nutrition or presence of inflammation affects serum albumin levels, we studied serum albumin levels, C-reactive protein (CRP) levels and caloric intake over time in 30 patients with burns. Serum albumin levels did not respond to provision of nutrition, but correlated negatively with CRP levels, suggesting that serum albumin levels are more closely associated with inflammation than nutrition. This study also suggests that hypoalbuminemia is a good indicator of the severity of burns or associated complications. We conclude that serum albumin levels do not offer a good nutritional marker in burns patients

Role of Sphingomyelinase in Infectious Diseases Caused by Bacillus cereus

Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H2O2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis

Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine

Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10

Clostridium perfringens Epsilon-Toxin Impairs the Barrier Function in MDCK Cell Monolayers in a Ca2+-Dependent Manner

Epsilon-toxin produced by Clostridium perfringens significantly contributes to the pathogeneses of enterotoxemia in ruminants and multiple sclerosis in humans. Epsilon-toxin forms a heptameric oligomer in the host cell membrane, promoting cell disruption. Here, we investigate the effect of epsilon-toxin on epithelial barrier functions. Epsilon-toxin impairs the barrier integrity of Madin-Darby Canine Kidney (MDCK) cells, as demonstrated by decreased transepithelial electrical resistance (TEER), increased paracellular flux marker permeability, and the decreased cellular localization of junctional proteins, such as occludin, ZO-1, and claudin-1. U73122, an endogenous phospholipase C (PLC) inhibitor, inhibited the decrease in TEER and the increase in the permeability of flux marker induced by epsilon-toxin. The application of epsilon-toxin to MDCK cells resulted in the biphasic formation of 1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). U73122 blocked the formation of DAG and IP3 induced by the toxin. Epsilon-toxin also specifically activated endogenous PLC-γ1. Epsilon-toxin dose-dependently increased the cytosolic calcium ion concentration ([Ca2+]i). The toxin-induced elevation of [Ca2+]i was inhibited by U73122. Cofilin is a key regulator of actin cytoskeleton turnover and tight-junction (TJ) permeability regulation. Epsilon-toxin caused cofilin dephosphorylation. These results demonstrate that epsilon-toxin induces Ca2+ influx through activating the phosphorylation of PLC-γ1 and then causes TJ opening accompanied by cofilin dephosphorylation

Delta-toxin caused mitochondrial dysfunction.

<p>(A) A549 cells preincubated with MitoTracker red and Hoechst 33342 were treated with delta-toxin (50 ng/ml) for 30 min at 37°C. (B) A549 cells were treated with delta-toxin (50 ng/ml) for 30 min at 37°C. Cells were formaldehyde-fixed, permeabilized and stained using an anti-cytochrome <i>c</i> antibody and Hoechst 33342. (C) A549 cells were treated with delta-toxin (50 ng/ml) for the indicated time periods at 37°C. The mitochondria and the cytosol fractions were prepared as described in the Materials and Methods, and then were subjected to immunoblotting for the detection of cytochorome <i>c</i>. (D,E) A549 cells transfected with Mito-GFP were treated with delta-toxin (50 ng/ml) for 30 min at 37°C. The cells were formaldehyde-fixed, permeabilized and stained with an active-form-specific anti-Bax antibody (D) or an active-form-specific anti-Bak antibody (E). Nuclear DNA was stained with Hoechst 33342. Cells were examined using a confocal microscope. These results are representative of four experimental studies. Bar, 5 μm.</p

Cytotoxic effect of delta-toxin on A549 cells.

<p>(A) A549 cells were treated with heat-inactivated (H.I.) delta-toxin (1 μg/ml) or delta-toxin (10 and 20 ng/ml) for the indicated time periods at 37°C. After washing, the cells were detached using trypsin, stained with propidium iodide (PI) and annexin V, treated for 15 min at 25°C and assessed by flow cytometry. Percentages of cells of each quadrant are shown in each dot-plot graph. Quantitative analysis of the staining is shown in (B). The values are the mean ±s tandard deviation (SD) of four experimental studies in each group. (C) A549 cells were incubated with delta-toxin (10, 20, and 50 ng/ml) at 37°C for the indicated time periods before lactate dehydrogenase (LDH) was measured. The means ± standard deviation (SD) of four independent experiments are shown.</p

Binding and oligomerization of delta-toxin in A549 cells.

<p>(A) A549 cells were treated with delta-toxin (1 μg/ml) for 30 min at 4°C. The cells were washed, and incubated for the indicated time periods at 37°C. The cells were dissolved in SDS-sample solution without heating and confirmed by immunoblotting of delta-toxin and β-actin (control). Representative data from one of four experimental studies are shown. (B) A549 cells were treated with delta-toxin for 1 h at 4°C or 37°C prior to an ATP assay. Results are indicated as percentage of the value for controls. The mean ± standard deviation (SD) for four experimental studies is shown. (C) SDS-PAGE analysis of purified delta-toxin. The purified preparation of delta-toxin (5 μg of protein) from cell lysates of <i>E</i>. <i>coli</i> transformants containing a delta-toxin encoding plasmid was resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie brilliant blue. Lanes: 1, molecular mass standards; 2, purified delta-toxin. (D) A549 cells were treated with Cy3-labeled delta-toxin (1 μg/ml) for 30 min at 4°C (panel a). After washing, cells were cultivated with medium for 30 min at 37°C (panel b). The cells were fixed using formaldehyde and stained with Hoechst 33342. Cy3-labeled delta-toxin (red) and the cell nuclei (blue) were examined using a confocal microscope. DIC, differential interference contrast. The images are representative of those from four experimental studies. Bar, 5 μm.</p

Delta-toxin binding to lipid rafts of A549 cells.

<p>(A) A549 cells were treated with delta-toxin (1 μg/ml) for 15 min at 37°C. After washing, the cells were dissolved in 1% Triton X-100. Then, lipid rafts were separated using a sucrose gradient. Portions of the 10 fractions from the gradient were assessed by immunoblotting using anti-delta-toxin, anti-caveolin-1, or anti-flotillin antibodies. The result is representative of four experimental studies. (B) Cholesterol distribution in the fractions of lipid rafts. Cholesterol distribution in lipid raft fractions was accessed as described in the Materials and Methods. (C) A549 cells were incubated with methyl-β-cyclodextrin (MβCD) for 60 min at 37°C. The cholesterol contents were assayed spectrophotometrically as described in the Materials and Methods. (D) A549 cells were treated with MβCD for 60 min at 37°C. The cells were treated with delta-toxin (50 ng/ml) for 30 min at 37°C prior to ATP assays. Data are reported as the percentage of the value for untreated controls. The mean ± standard deviation (SD) of four experimental studies is shown. Results were assessed using one-way ANOVA with Bonferroni’s multiple-comparison post-test. *<i>P</i><0.05, **<i>P</i><0.01. (E) A549 cells were incubated with MβCD for 60 min at 37°C. The cells were treated with delta-toxin (1 μg/ml) for 30 min at 37°C. The cells were solubilized in SDS-sample solution and confirmed by immunoblotting using anti-delta-toxin or anti-β-actin antibodies. The result is representative of four experimental studies. (F) Partial colocalization of GM1-rich rafts and delta-toxin. A549 cells were treated with Cy3-labeled delta-toxin (1 μg/ml) and Alexa Fluor 488-labeled cholera toxin B subunit (CTB) (1 μg/ml) for 15 min at 37°C. Delta-toxin (red) and CTB (green) were examined using a confocal microscope. The arrowhead (yellow) shows the colocalization of delta-toxin with CTB. The result is representative of four experimental studies. Bar, 5 μm.</p

Delta-toxin induced carboxyfluorescein release from phospholipid-cholesterol liposomes.

<p>(A) Carboxyfluorescein (CF)-loaded liposomes each composed of SM or PC and cholesterol at a molar ratio of 50:50 mol % were treated with delta-toxin for 30 min at 37°C. (B) Liposomes composed of sphingomyelin (SM) and cholesterol at several molar ratios were treated with delta-toxin for 30 min at 37°C. The molar ratio of cholesterol to SM (mol %) was 50, 40, or 30. CF release was measured as described in the Materials and Methods. The mean ± standard deviation (SD) of four experimental studies is shown. (C) Liposomes composed of SM and cholesterol at various molar ratios (50, 40 or 30 mol %) were treated with delta-toxin (1 μg/ml) for 30 min at 37°C. Liposome-bound toxin was solubilized and confirmed by immunoblotting of delta-toxin. The result is representative of four experimental studies. (D) Effect of cholesterol on cytotoxicity caused by delta-toxin. To assay cholesterol inhibition, a 50 μl volume of cholesterol in absolute ethanol was added to 1 ml aliquots of 50 ng/ml delta-toxin preparations, to a final concentration of 1 μg/ml. After 30 min treatment at room temperature, cytotoxicity was assayed as described in the Materials and Methods. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazilum (MTS) method. Results are indicated as percentage of the value for controls. The mean ± standard deviation (SD) for four experimental studies is shown.</p