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Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Previously, rodent and human monocytes/macrophages killed glioma cells bearing the membrane macrophage colony stimulating factor (mM-CSF) through paraptosis, but the molecular mechanism of this killing process was never identified. We have demonstrated that paraptosis of rat T9 glioma cells can be initiated through a large potassium channel (BK)-dependent process initiated by reactive oxygen species. Macrophage mediated cytotoxicity upon the mM-CSF expressing T9-C2 cells was not prevented by the addition of the caspase inhibitor, zVAD-fmk. By a combination of fluorescent confocal and electron microscopy, flow cytometry, electrophysiology, pharmacology, and genetic knock-down approaches, we demonstrated that these ion channels control cellular swelling and vacuolization of rat T9 glioma cells. Cell lysis is preceded by a depletion of intracellular ATP. Six-hour exposure to BK channel activation caused T9 cells to over express heat shock proteins (Hsp 60, 70, 90 and gp96). This same treatment forced HMGB1 translocation from the nuclear region to the periphery. These last molecules are “danger signals” that can stimulate immune responses. Similar inductions of mitochondrial swelling and increased Hsp70 and 90 expressions by BK channel activation were observed with the non-immunogenic F98 glioma cells. Rats injected with T9 cells which were killed by prolonged BK channel activation developed immunity against the T9 cells, while the injection of x-irradiated apoptotic T9 cells failed to produce the vaccinating effect. These results are the first to show that glioma cellular death induced by prolonged BK channel activation improves tumor immunogenicity; this treatment reproduces the vaccinating effects of mM-CSF transduced cells. Elucidation of strategies as described in this study may prove quite valuable in the development of clinical immunotherapy against cancer.

Human U251MG glioma cells expressing the membrane form of macrophage colony-stimulating factor (mM-CSF) are killed by human monocytes in vitro and are rejected within immunodeficient mice via paraptosis that is associated with increased expression of three different heat shock proteins Martin R Jadus, Human U251MG glioma cells retrovirally transduced with the human gene for the membrane form of macrophage colonystimulating factor (mM-CSF) were investigated. The clones, MG-2F11 and MG-2C4, that expressed the most mM-CSF, but not the viral vector or the parental U251MG cells, were killed by both murine and human monocyte/macrophages in cytotoxicity assays. MG-2F11 cells failed to form subcutaneous tumors in either nude or NIH-bg-nu-xidBR mice, while mice inoculated with the U251MG viral vector (MG-VV) cells developed tumors. Electron microscopy studies showed that 4 hours after subcutaneous injection, the mM-CSF-transduced cells began dying of a process that resembled paraptosis. The dying tumor cells were swollen and had extensive vacuolization of their mitochondria and endoplasm reticulum. This killing process was complete within 24 hours. Macrophage-like cells were immediately adjacent to the killed MG-2F11 cells. Immunohistological staining for the heat shock proteins HSP60, HSP70 and GRP94 (gp96) showed that 18 hours after inoculation into nude mice, the MG-2F11 injection site was two to four times more intensely stained than the MG-VV cells. This study shows that human gliomas transduced with mM-CSF have the potential to be used as a safe live tumor cell vaccine.

Cryptococcus neoformans is an opportunistic fungal pathogen that synthesizes and catabolizes inositol. This study demonstrates inositol synthesis from glucose‐6‐phosphate via inositol‐1‐phosphate synthase and catabolism to glucuronic acid via inositol oxygenase in this organism. These inositol synthetic and catabolic pathways are regulated in opposition; repressing conditions for one are inducing conditions for the other. An inositol‐requiring strain was generated by UV mutagenesis. Without inositol, this mutant strain undergoes ‘inositol‐less’ death, during which time the phosphatidylinositol composition of the membranes decreases without alteration of the proportion of other phospholipids. The mutation on this strain results in no detectable inositol synthetic activity but normal (wild‐type) inositol catabolic activity. This inositol‐requiring mutant strain reverted at a high frequency. Classical genetic experiments revealed that the majority of the reverting mutations are at second sites. Interestingly, the revertants exhibited unusual morphological phenotypes when deprived of inositol, while provision of inositol restored wild‐type morphology. Inositol metabolism is clearly important for growth and development of C. neoformans and may be involved in this organism's mechanism for survival as both a saprophyte in soil and a parasite in humans. Copyright © 1999 John Wiley & Sons, Ltd.

It has long been assumed that the mucosa in areas of grossly 'healed' gastric or duodenal ulcers returns to normal, either spontaneously or after treatment. This assumption is based almost entirely upon visual, superficial examination by endoscopy. Few, if any, histological and ultrastructural studies examined the deeper mucosa in the areas of grossly healed ulcers. In several experimental studies, we analysed the development, evolution, and healing of acetic acid-induced gastric ulcers in rats and assessed the histological and ultrastructural features (structure and cellular composition) of the gastric mucosa in areas of grossly healed ulcers. The gastric mucosa of grossly 'healed' ulcers showed re-epithelialization of the mucosal surface at every study interval (2 weeks, 2, 3, and 4 months), but the subepithelial mucosa displayed prominent abnormalities. Two patterns of scarring were distinguished : (a) the mucosa in the area of healed ulcer was thinner (25-45 % thinner than normal mucosa) with increased connective tissue and poor differentiation and/or degenerative changes in the glandular cells; and (b) the mucosa displayed a marked dilation of gastric glands with poor differentiation of

The development of Sarcocystis cruzi Hasselmann (syn. S. fusiformis Railliet) meronts was studied in seven 7- to 10-day-old calves filled 4, 7, 11, 15, 22, 25 and 28 days postinoculation (DPI) with 5 x 10(7) sporocysts from feces of coyotes. No meronts were found 4 and 7 DPI. Young and intermediate meronts with 1-16 nuclei were found in endothelial cells of arteries in mesenteric lymph nodes, but not in kidneys 11 DPI. Mature meronts were noted in endothelial cells of arteries, arterioles, or capillaries of many organs of calves killed 15 to 25 DPI. No first-generation meronts were found 28 DPI. By electron microscopy, all stages of the first-generation merogony were found free within the host cell cytoplasm and not within a parasitophorous vacuole. The appearance of intranuclear spindles preceded the formation of merozoites by endopolygeny. Mature meronts measured 41.0 x 17.5 (34-50 x 15-24) microgram, contained approximately 100-350 merozoites, and had 2 to 4 relatively small residual bodies, 2.8 microgram in diameter. Merozoites measured 6.3 x 1.5 (5.5-7 x 1 microgram) and contained most of the organelles characteristically found in coccidian merozoites. Micropores were observed in merozoites, but not in young and intermediate meronts. Merozoites were seen free in the lumen of blood vessels, in intracellular areas, and free within the host cell cytoplasm.