To determine if megakaryocytes are targeted by immune thrombocytopenic purpura (ITP) autoantibodies, as are platelets, we have studied the effects of ITP plasma on in vitro megakaryocytopoiesis. Umbilical cord blood mononuclear cells were incubated in the presence of thrombopoietin and 10% plasma from either ITP patients (n = 53) or healthy donors. The yield of megakaryocytic cells, as determined by flow cytometry, was significantly reduced in the presence of ITP plasma containing antiplatelet glycoprotein Ib (GPIb) autoantibodies (P < .001) as compared with both the control and patient plasma with no detectable anti-GPIIb/IIIa or anti-GPIb autoantibodies. Platelet absorption of anti-GPIb autoantibodies in ITP plasmas resulted in double the megakaryocyte production of the same plasmas without absorption, whereas platelet absorption of control plasma had no effect on megakaryocyte yield. Furthermore, 2 human monoclonal autoantibodies isolated from ITP patients, 2E7, specific for human platelet glycoprotein IIb heavy chain, and 5E5, specific for a neoantigen on glycoprotein IIIa expressed on activated platelets, had significant inhibitory effects on in vitro megakaryocytopoiesis (P < .001). Taken together, these data indicate that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.

A biphasic plasmid vector was used to introduce the Escherichia coli K-12 lac operon into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6. The PR-6 transformants expressed β-galactosidase at nearly as high a level as did Escherichia coli transformants. In order to accomplish this, it was necessary to obtain PR-6 mutants that could be transformed by plasmids with unmodified recognition sites for the endogenous PR-6 restriction endonuclease Aqu I. These mutants were generated by a variation of the ectopic mutagenesis techniques that have been used in other naturally transforming bacteria. The ability to assay the expression of lacZ in PR-6 paves the way for the construction of gene fusions with various PR-6 promoters and quantitation of their expression under specific in vivo conditions.

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t ) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3′-untranslated region (3′-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitrodecay of the GM-CSF 3′-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3′-UTR are generated that are truncated at either end of the ARE. Furthermore, thet of the ARE-containing 3′-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3′-UTRin vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.