Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

Apoptosis of human T-cell acute lymphoblastic leukemia cells by diphenhydramine, an H1 histamine receptor antagonist.

Recently, it has been demonstrated that histamine plays an important role in the proliferation of normal and malignant cells. We have examined the effects of histamine, diphenhydramine, and cimetidine (H1 and H2 histamine receptor antagonists, respectively) on the in vitro proliferation of two human T-cell acute lymphoblastic leukemia cell lines, namely CCRF-CEM and Jurkat. Exogenous histamine did not alter the proliferation or viability of these cells. In contrast, diphenhydramine induced apoptosis in a dose- and time-dependent manner in both cell lines, whereas cimetidine failed to induce significant effects at similar concentrations. Diphenhydramine-induced apoptosis was evaluated in terms of morphology, flow cytometry, and the release of cytochrome c to the cytosol. The latter was partially mitigated by Bcl-2 overexpression. In human peripheral blood mononuclear cells, diphenhydramine inhibited cell proliferation without inducing apoptosis. Our findings indicate that endogenous histamine may be an important factor for the survival of CCRF-CEM, Jurkat, and peripheral blood mononuclear cells, and point to the potential application of H1 receptor antagonists as cytotoxic agents for the specific treatment of certain types of leukemia.

Localization of importin alpha (Rch1) at the plasma membrane and subcellular redistribution during lymphocyte activation.

Rch1 belongs to the importin alpha subfamily and works as an adapter between karyophilic proteins and the nuclear import machinery. Its level of expression varies among species and tissues, and depends on the state of cellular metabolism. In the present study we examined the level of expression of nuclear envelope and nuclear transport proteins (Rch1, importin beta, lamins A/C, lamin B, gp210, p62 and transportin) after human lymphocyte activation with phytohemagglutinin. We observed that the level of Rch1 increases dramatically, especially in larger lymphocytes, in response to activation. Moreover, using immunoelectron microscopy, this nuclear transport factor was found to be localized at the plasma membrane and also in tracks from the cytoplasm through the nuclear envelope into the nucleus. Similar localization was also observed in the human melanoma cell line A375. In addition, metabolic activation led to a redistribution of Rch1 from the cytoplasm to both the plasma membrane and the nuclear interior. These results suggest that, during lymphocyte activation, Rch1 may be involved in a signal transduction pathway that involves the shuttling of karyophilic proteins from the plasma membrane to the nucleus.