Early/memory and plasma B-cell lines and fibroblasts were analyzed for their ability to use a 5' proximal (variant) versus a 3' distal (constant) poly(A) site, in the absence of a competing splice, from a set of related constructs. The proximal:distal poly(A) site use (P:D ratio) of the resulting cytoplasmic poly(A)+ mRNA is a measure of poly(A) site strength. In this context the immunoglobulin gamma2b secretory-specific poly(A) site showed a P:D ratio of 1:1 in plasma cells, 0.43:1 in early/memory B-cells and an intermediate value in fibroblasts. Meanwhile, a construct with a proximal SV40 early-like poly(A) site produced mRNA with a P:D ratio of >>50:1 in all cell types. Alterations in the region downstream of the proximal poly(A) addition site and at the site itself resulted in changes in the P:D ratio. However, these poly(A) sites, all with a P:D ratio of < or = 5:1, were used most efficiently in plasma cells. Constructs totally devoid of immunoglobulin sequences, but containing heterologous poly(A) sites producing mRNA with P:D ratios of < or = 5:1, were also used more efficiently in plasma cells. We therefore conclude that weak poly(A) sites, regardless of sequence composition, are used more efficiently in plasma cells than in the other cell types.
Liver transplantation k a well-established therapeutic option forpatients with end-stage liver dkease. However, up to 20% of transplanted livers fail to have adequate function initially, and at least harf of those will eventually fail. Accurate, early prediction of outcome may ameliorate thk situation by encouraging retransplantation before the patient's condition becomes irreversible.In thk shrdy, clinical information was gathered prospectively for 295 patients who underwent liver transplantation at the Universiy of Pittsburgh Medical Center, and was divided into sets. The feed-forward, fully connected, neural networks had 7 or 8 inputs, a single hidden layer conskting of 3 nodes and a single output node cfailure=I, success=O). The networks were trained with data from a randomly selected subset of 240 patients while the remaining 55 patients made up the test set. The preoperative (day 0) data conskted of patient demographics plus the results of standard liver function tests. The "day 1" data consisted information gathered during surgery plus the prediction of outcome from day 0. Data for days 2-5 included resultsfrom standard liver function tests plus the prediction of outcome from the previous day's network The network was trained using a standard back propagation algorithm. naining was assessed by testing the abiliy of the network to correctly predict the outcome of the 55 patients in the test set. The accuracy of prediction by the neural network improved each day and so by day 5, 98% of the grafi survivors in the test set were correctly predicted while 88% of grafi failures in the test set were correctly predicted.
To determine the role of natural killer (NK) cells in the regulation of human erythropoiesis, we studied the effects of NK-enriched cell populations on the in vitro proliferation of erythroid stem cells at three different levels of maturation (day 14 blood BFU-E, day 5–6 marrow CFU-E, and day 10–12 marrow BFU-E). NK cells were enriched from blood by Percoll density gradient centrifugation and by fluorescence- activated cell sorting (FACS), using the human natural killer cell monoclonal antibody, HNK-1. The isolated enriched fractions were cocultured with autologous nonadherent marrow cells or blood null cells and erythropoietin in a methylcellulose erythroid culture system. Cells from low-density Percoll fractions (NK-enriched cells) were predominantly large granular lymphocytes with cytotoxic activity against K562 targets 6–10-fold greater than cells obtained from high- density Percoll fractions (NK-depleted cells). In coculture with marrow nonadherent cells (NA) at NK:NA ratios of 2:1, NK-enriched cells suppressed day 5–6 CFU-E to 62% (p less than 0.025) of controls, whereas NK-depleted cells slightly augmented CFU-E to 130% of controls (p greater than 0.05). In contrast, no suppression of day 10–12 marrow BFU-E was observed employing NK-enriched cells. The NK CFU-E suppressor effects were abolished by complement-mediated lysis of NK-enriched cells with the natural killer cell antibody, HNK-1. Highly purified HNK- 1+ cells separated by FACS suppressed marrow CFU-E to 34% (p less than 0.025) and marrow BFU-E to 41% (p less than 0.025) of controls. HNK- cells had no significant effect on either BFU-E or CFU-E growth. NK- enriched cells were poor stimulators of day 14 blood BFU-E in comparison to equal numbers of NK-depleted cells or T cells isolated by E-rosetting (p less than 0.01). Interferon boosting of NK-enriched cells abolished their suboptimal burst-promoting effects and augmented their CFU-E suppressor effects. These studies provide evidence for a potential regulatory role of NK cells in erythropoiesis. The NK suppressor effect is maximal at the level of the mature erythroid stem cell CFU-E. These findings may explain some hypoproliferative anemias that develop in certain NK cell-activated states.
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