The experimental infection of rats with seven strains of Entamoeba histolytica were carried out according to animal ages, number of inoculated amebae, rat strain differences and rat-culture passages. The rat cecal scoring technique of Neal (1951) was utilized to measure the invasiveness of the parisite. The results are summarized and concluded as follows; 1. In the infection of Sprague-Dawley strain rat with YS9-strain and NAMRU II-stran amebae, which wem confirmed highly invasive to the membrane of cecum of rabbits in the previous reports (Cho,1968; Cross,1968), remarkab1e invasiveness was observed in the 30-day-old rat groups with the average cecal score above 5.0. Although no statistical differences of virulence by the number of inoculations showed in rat groups, the cecal scores were markedly reduced in the 50,000 amebae inoculated rats. 2. The hybrid albino rats were considered unsuitable for virulence study of E. histolytica, since the invasiveness of the amabae was inconsistant. 3. The virulences of YS 14 and YS16-strains from cyst carrier showed no virulence, YS 15 from cyst carrier and YS 24 from liver abscess were moderately invasive, and only YS25 from liver abscess showed highly invasive as with YS 9 and NAMRU II-strain amebae. By rat-culture passage, YS14-strain and YS24-strain amebae showed marked increase of invasiveness. It was presumed that the rat-culture passage should be indispensably supplememed in the studies on the virulence of E. histolytica.
Adult ; Animals ; Cecum/microbiology ; Entamoeba histolytica/*pathogenicity ; Female ; Human ; Korea ; Male ; Middle Aged ; Rats ; Virulence

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and micro-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and micro-calpain may be involved in colon epithelial cell death induced by E. histolytica.
Calpain/antagonists & inhibitors/genetics/*metabolism ; *Cell Death ; Cell Line ; Cell Survival/drug effects ; Dipeptides/metabolism ; Entamoeba histolytica/*pathogenicity ; Epithelial Cells/*parasitology ; Gene Knockdown Techniques ; Humans

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.
*Cell Death ; Cell Line ; Entamoeba histolytica/*pathogenicity ; Epithelial Cells/metabolism/*parasitology/*physiology ; *Host-Pathogen Interactions ; Humans ; NADPH Oxidase/*metabolism ; Reactive Oxygen Species/metabolism/*toxicity ; Signal Transduction

Tyrosine kinases are one of the most important regulators for intracellular signal transduction related to inflammatory responses. However, there are no reports describing the effects of tyrosine kinases on neutrophil apoptosis induced by Entamoeba histolytica. In this study, isolated human neutrophils from peripheral blood were incubated with live trophozoites in the presence or absence of tyrosine kinase inhibitors. Entamoeba-induced receptor shedding of CD16 and PS externalization in neutrophils were inhibited by pre-incubation of neutrophils with the broad-spectrum tyrosine kinase inhibitor genistein or the Src family kinase inhibitor PP2. Entamoeba-induced ROS production was also inhibited by genistein or PP2. Moreover, genistein and PP2 blocked the phosphorylation of ERK and p38 MAPK in neutrophils induced by E. histolytica. These results suggest that Src tyrosine kinases may participate in the signaling event for ROS-dependent activation of MAPKs during neutrophil apoptosis induced by E. histolytica.
*Apoptosis ; Cells, Cultured ; Entamoeba histolytica/*immunology/*pathogenicity ; GPI-Linked Proteins/metabolism ; Genistein/metabolism ; Humans ; Neutrophils/*immunology ; Protein Kinase Inhibitors/metabolism ; Pyrimidines/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, IgG/metabolism ; src-Family Kinases/antagonists & inhibitors/*metabolism