Lymphokine synthesis patterns of a panel of 19 T cell clones have been evaluated, using mRNA hybridization methods to examine 11 different mRNAs induced by Con A. The two types of CD4+ Th cell clone described previously were clearly distinguished by this procedure, and the differences between the two types have now been extended to six induced products. With minor exceptions, only Th1 clones synthesized mRNA for IL-2, IFN-gamma, and lymphotoxin, and only Th2 clones synthesized mRNA for IL-4, IL-5, and another induced gene, P600. Four more induced products were expressed preferentially but not uniquely by one or another type of clone: mRNAs for GM-CSF, TNF, and another induced, secreted product (TY5) were produced in larger amounts by Th1 clones, whereas preproenkephalin was preferentially expressed by Th2 clones. IL-3 was produced in similar amounts by both types of clone. mAbs were used to establish three bioassays that were functionally monospecific for IL-2, IL-3, and IL-4, and a new anti-IFN gamma mAb, XMG1.2, was used to establish an ELISA for IFN-gamma. These four assays were used to show that secreted protein and mRNA levels correlated well for all cell lines. The implications of these findings for normal T cells are discussed.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.

Apicomplexan parasites secrete transmembrane (TM) adhesive proteins as part of the process leading to host cell attachment and invasion. These microneme proteins are cleaved in their TM domains by an unidentified protease termed microneme protein protease 1 (MPP1). The cleavage site sequence (IAYGG), mapped in the Toxoplasma gondii microneme proteins TgMIC2 and TgMIC6, is conserved in microneme proteins of other apicomplexans including Plasmodium species. We report here the characterisation of novel T. gondii proteins belonging to the rhomboid family of intramembrane-cleaving serine proteases. T. gondii possesses six genes encoding rhomboid-like proteins. Four are localised along the secretory pathway and therefore constitute possible candidates for MPP1 activity. Toxoplasma rhomboids TgROM1, TgROM2 and TgROM5 cleave the TM domain of Drosophila Spitz, an established substrate for rhomboids from several species, demonstrating that they are active proteases. In addition, TgROM2 cleaves chimeric proteins that contain the TM domains of TgMIC2 and TgMIC12. q

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The RIE-1 cell line is an untransformed, epithelial cell line derived from the rat small intestine. We report that epidermal growth factor (EGF), which regulates the proliferation of RIE-1 cells, also directs their movement. We measured cell migration through gelatin-coated filters in blind-well Boyden chambers. The migration of RIE-1 cells was stimulated up to approximately 100-fold by EGF, with a half-maximal response at 1-2 ng/ml and a maximal effect at 10 ng/ml. Further analysis showed that the RIE-1 cells responded directionally to a gradient of EGF in solution. Other growth factors tested did not stimulate RIE-1 cell migration, and EGF did not stimulate the migration of fibroblasts in this assay. We conclude that EGF is a potent and specific chemo-attractant for RIE-1 intestinal epithelial cells and suggest that EGF might influence epithelial cell migration in vivo.

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Background Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species. Methods Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro–computed tomography and histology. Results Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells. Conclusions Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.