Bacterial lipopolysaccharide induces cytoskeletal rearrangement in small intestinal lamina propria fibroblasts: actin assembly is essential for lipopolysaccharide signaling.

Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on the dynamics and organization of the cytoskeletal proteins, actin, vimentin, tubulin and vinculin in human small intestinal lamina propria fibroblasts (HSILPF). A noticeable change in the actin architecture was observed after 30 min incubation with LPS with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 2 h. Reorganization of the vimentin network into vimentin bundling was conspicuous at 2 h. With further increase in the time period of LPS exposure, diffused staining of vimentin along with vimentin bundling was observed. Vinculin plaques distributed in the cell body and cell periphery in the control cells rearrange to cell periphery in LPS-treated cells by 30 min of LPS exposure. However, there was no change in the tubulin architecture in HSILPF in response to LPS. LPS increased the F-actin pool in HSILPF in a concentration-dependent manner with no difference in the level of G-actin. A time-dependent study depicted an increase in the G-actin pool at 10 and 20 min of LPS exposure followed by a decrease at further time intervals. The F-actin pool in LPS-treated cells was lower than the control levels at 10 and 20 min of LPS exposure followed by a sharp increase until 120 min and finally returning to the basal level at 140 and 160 min. Further (35)S-methionine incorporation studies suggested a new pool of actin synthesis, whereas the synthesis of other cytoskeletal filaments was not altered. Cytochalasin B, an actin-disrupting agent, severely affected the LPS induced increased percentage of 'S' phase cells and IL-6 synthesis in HSILPF. We conclude that dynamic and orchestrated organization of the cytoskeletal filaments and actin assembly in response to LPS may be a prime requirement for the LPS induced increase in percentage of 'S' phase cells and IL-6 synthesis

Microplate assay for detection of bacterial contamination in tissue culture media.

In the present study, a rapid and simple colorimetric technique has been described to determine the presence of bacteria in tissue culture medium used in animal cell culture. The microplate assay is based on utilization of MTT [3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] by bacteria resulting in formation of formazan crystals which can be measured colorimetrically. Contaminated medium, a standard gram-negative and gram-positive bacteria produce formazan from MTT which is related to the bacterial load. The assay has utility in screening tissue culture reagents to detect the presence of bacteria.

Adherence of Streptococcus pyogenes M type 5 to pharyngeal and buccal cells of patients with rheumatic fever and rheumatic heart disease during a one-year follow-up.

In vitro adherence of Streptococcus pyogenes M type 5 to isolated pharyngeal and buccal epithelial cells was studied in patients with acute recurrent rheumatic fever (n = 21), chronic rheumatic heart disease (n = 33), streptococcal pharyngitis (n = 12), and in normal controls. Patients were investigated at admission and one, six and 12 months later. Streptococci adhered significantly more to the pharyngeal cells of patients with rheumatic fever and rheumatic heart disease than to the pharyngeal cells of controls. Adherence of streptococci to pharyngeal cells of patients with pharyngitis was not different from age-matched controls. The adherence of streptococci to the pharyngeal cells of patients with acute rheumatic fever fell during follow-up but even after one year remained significantly higher than in the control group. These findings suggest that host factor(s) controlling streptococcal adhesion and colonization at the pharyngeal mucosa may be important in the pathogenesis of acute rheumatic fever.