Kathryn E. Bass

Maternal smoking inhibits early human cytotrophoblast differentiation

Differentiation of the specialized epithelial cells of the placenta, termed cytotrophoblasts, is a particularly important aspect of placental development during the first trimester of pregnancy. During this process cytotrophoblast stem cells either fuse to form the syncytium or aggregate to form cell columns that adhere to, then invade the uterus. We found that chorionic villi from early gestation placentas of mothers who smoke showed a marked reduction in cell columns, a defect that could not be corrected by placing them in culture. We used two different in vitro models to determine if nicotine plays a role in the etiology of this defect. Exposing early gestation chorionic villi from nonsmoking women to nicotine inhibited subsequent cell column formation in vitro. Nicotine also inhibited normal first trimester cytotrophoblast invasion, apparently by reducing the ability of treated cells to synthesize and activate the 92 kDa type IV collagenase, an important mediator of invasion in vitro. These results suggest that maternal cigarette smoking inhibits the trophoblast differentiation pathway that leads to column formation and uterine invasion. This effect, which is due at least in part to the effects of nicotine, may contribute to the growth retardation observed in fetuses of mothers who smoke during pregnancy.

The in vitro differentiation of pleomorphic Trypanosoma brucei from bloodstream into procyclic form requires neither intermediary nor short-stumpy stage.

Our studies on the in vitro differentiation of a pleomorphic Trypanosoma brucei strain TREU667 indicate that the parasite differentiates directly from long-slender into procyclic form when incubated in Cunninghams medium at 26 degrees C. The intermediary and the short-stumpy bloodstream forms harvested from infected mice can also differentiate directly into procylic form in vitro with time courses similar to that for the long-slender form. Thus, none of the three bloodstream forms appear to be significantly better preadapted for differentiation. Tricarboxylic acid (TCA) cycle intermediates cis-aconitate and L-citrate can shorten the initial lag phase of the differentiation, but an essential trigger appears to be the temperature shift from 37 degrees C to 26 degrees C, when other TCA cycle intermediates such as L-proline, L-malate, alpha-ketoglutarate, fumarate and succinate are present in Cunninghams medium. The ornithine decarboxylase (ODC) activity in T. brucei showed a gradual increase and the ODC mRNA level remained constant during the differentiation. DL-alpha-Difluoromethylornithine (DFMO), putrescine, dibutyryl cAMP and theophylline all exerted no discernible effect on the in vitro process, which suggests that neither cAMP increase nor polyamine depletion could be counted among the triggers of T. brucei differentiation. A monomorphic T. brucei strain EATRO110 was tested in the same medium at 26 degrees C but was unable to differentiate.

Human trophoblast invasion. Autocrine control and paracrine modulation.

Development of the human placenta involves rapid invasion of the uterine wall by fetal trophoblasts, a process with certain similarities to tumor cell invasion. Unlike tumor invasion, however, this unique interaction between genetically dissimilar trophoblast and uterine cells is closely regulated and limited both temporally and spatially by mechanisms that are largely unknown. We have used a combination of two experimental approaches to study this process: immunolocalization using tissue sections to investigate trophoblast invasion in vivo, and a cell culture model that allows manipulation of the invasion process in vitro. The results show that invading trophoblasts express activated forms of metalloproteinases, adhesion molecules and the novel class I histocompatibility antigen, HLA-G, in a highly regulated manner during invasion. The behavior of cytotrophoblasts in vitro, removed from the influences of uterine cells, closely parallels their behavior in vivo, suggesting the existence of autocrine control mechanisms. However, studies examining the effect of growth factors and cytokines on trophoblast invasion suggest that molecules of uterine origin can modify this process. Thus, we hypothesize that the intrinsic invasiveness of these cells is controlled, at least in part, by the specialized environment of the uterus. Future studies will concentrate on identifying these factors and the specific trophoblast functions they modify.

Transient inhibition of protein synthesis accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms

It has been widely believed that bloodstream forms of Trypanosoma brucei must be first transformed into intermediary and/or short-stumpy forms in the bloodstream of the mammalian host before differentiation to the procyclic culture form can occur. In our recent studies, the pleomorphic T. brucei strain TREU667 was found to differentiate directly from the long-slender bloodstream form to the procyclic form in Cunninghams medium at 26 degrees C [7]. In the present investigation, the same was found true for another pleomorphic strain of T. brucei, STIB366D. Four independent monomorphic strains of T. brucei were tested; two, #427 and EATRO164, were found capable of differentiating in vitro directly into procyclic forms, whereas the other two, TREU667/RP-56 and EATRO110, could not. There is thus no correlation between the capability of differentiating in vitro and the ability of being converted from long-slender to intermediary and short-stumpy bloodstream forms. Two additional markers for following differentiation, other than observing morphological changes, were tested. Assays for the emerging phosphoenolpyruvate carboxykinase (PEPCK) by immunoblottings worked well, with results agreeing closely with the morphological change. But immunoblottings of glycosomal phosphoglycerate kinase (gPGK) failed to demonstrate a significant decrease in the protein level upon completion of differentiation. Apparently, gPGK has a rather long half-life and is unsuitable as a marker of differentiation. When temperature was dropped from 37 degrees C to 26 degrees C at the starting point of in vitro differentiation, protein synthetic activity in the pleomorphic T. brucei TREU667 bloodstream form was decreased by 4-fold. When the activity was gradually brought back to and beyond the original level after a days incubation, the profile of newly synthesized proteins was that of the procyclic form. A monomorphic variant of TREU667, RP-56, which is incapable of differentiating in vitro, has a much higher protein synthetic activity than its pleomorphic parent in the bloodstream form. This high activity and the bloodstream profile of proteins thus synthesized were unaffected by the decreased temperature in Cunninghams medium until cell death. We thus conclude that a general inhibition of protein synthesis in bloodstream forms caused by temperature drop may be among the early events triggering differentiation into the procyclic form.

Regulation of Human Cytotrophoblast Invasion

Human cytotrophoblast invasion of the uterus is the result of an unusual differentiation process in which polarized epithelial cells, anchored to the chorionic villus basement membrane, become detached, aggregate into multilayered columns of nonpolarized cells, and rapidly penetrate the endometrium, the first third of the myometrium, and the associated spiral arterioles (Fig. 12.1). This process continues through the first trimester, peaks during the 12th week of pregnancy, and declines rapidly thereafter (1–4). The result is formation of the hemochorial placenta in which the fetal trophoblast cells are constantly bathed by maternal blood.

Epidermal Growth Factor Up-Regulates Human Cytotrophoblast Invasion

Successful development of the human fetus is contingent on the formation of cytotrophoblast cell columns that anchor the placenta to the uterus. Cytotrophoblasts, the specialized epithelial cells of the placenta, have the ability to differentiate along one of two pathways. In one pathway cytotrophoblasts detach from their basement membrane and fuse to form the multinucleate syncytium covering the floating chorionic villi. These cells are in direct contact with maternal blood and function in gas and nutrient exchange for the fetus. In the other pathway cytotrophoblasts also detach from their basement membrane and then aggregate to form the anchoring villi, multilayered columns of individual cells that adhere to the uterus. Cytotrophoblasts in the columns ultimately invade the decidua, the inner third of the myometrium, and the arterioles that supply these areas.