Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-μm-thick sections was used to detect bacteria after injection of ∼100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

The presence of the extracellular calcium-sensing receptor on human antral gastrin cells was investigated. Reverse transcription PCR using mRNA isolated from gastrin cell- enriched cell cultures identified a product with a sequence identical to part of the human parathyroid-secreting cell calcium-sensing receptor. Immunocytochemistry with an antibody to the extracellular region of the receptor immunostained all gastrin cells (but not mucin or somatostatin cells), and detected appropriate-sized bands in Western blots of whole cell lysates. Increasing extracellular calcium levels from 0.5 to 9 mM stimulated gastrin release in a concentration-dependent manner, with maximal release obtained at 7.2 mM. A known agonist of the calcium receptor, spermine also stimulated gastrin release. Microfluorimetry of identified gastrin cells demonstrated that increasing extracellular calcium resulted in an initial rapid rise in intracellular calcium followed by a plateau level that returned to basal levels immediately after removal of the elevated calcium. The traces were consistent with activation of a receptor-mediated mechanism rather than a concentration-dependent influx of calcium. In conclusion, these data indicate that G cells express the calcium-sensing receptor, and that activation of the receptor may explain the acid rebound phenomenon associated with calcium-containing antacid preparations.