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 antiGPIIb/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. (Blood. 2003;102: 887-895)

No abstract

The regulation of megakaryocytopoiesis and thrombopoiesis appears to be under the control of an array of hematopoietic growth factors. To determine the relationship of endogenous thrombopoietic cytokine levels and circulating platelet (PLT) counts, we measured the levels of thrombo-poietin (TPO), interleukin-11 (IL-11), and interleukin-6 (IL-6) in patients with significant thrombocytopenia secondary to both marrow hypoplasia and increased PLT destruction. Increased endogenous levels of TPO and IL-11, but not IL-6, were detected in bone marrow transplant patients with thrombocytopenia following myeloablative therapy (BMT/MAT) (TPO: 1,455.5 +/- 87.3 pg/mL, [PLT 39,600 +/- 7,800/microL], P < .001, n = 12; IL-11: 227.9 +/- 35 pg/mL, [PLT 32,900 +/- 57,000/microL], P < .05, n = 19; IL-6: 25.8 +/- 8.4 pg/mL, [PLT 32,800 +/- 5,057/microL], P > .05, n = 4] v normal donors [TPO < 150 pg/mL, n = 8; IL-11 < 50 pg/mL, n = 9; IL-6 < 10 pg/mL, n = 5 [PLT 203,000 +/- 7,500/microL]. There was a significant inverse correlation between endogenous levels of TPO and IL-11, but not IL-6, and PLT counts in the MAT/BMT patients (TPO: r = -0.57, P < .0001, n = 188; IL-11: r = -0.329, P < .0001, n = 249; IL-6: r = -0.1147, P > .05, n = 62). In patients with immune thrombocytopenia purpura (ITP), with decreased PLT survival, but intact bone marrow megakaryocytopoiesis, endogenous IL-11 levels were significantly increased (328.0 +/- 92.6 pg/mL, [PLT: 20,900 +/- 3,000/microL], P < .05, n = 25). However, endogenous TPO levels remained undetectable (< 150 pg/mL, [PLT 30,500 +/- 5,500/microL], n = 15). These results suggest that there may be differential mechanisms regulating endogenous TPO, IL-11, and IL-6 levels during acute thrombocytopenia and suggest that the absolute number of circulating PLTs may not always be the sole regulator of endogenous TPO levels. Other mpl-expressing cells of the megakaryocyte lineage may contribute to the regulation of circulating TPO levels as well. Our results also suggest IL-11 levels may in part, be regulated by a negative feedback loop based on circulating PLT counts, but also may, in part, be regulated by a variety of inflammatory agonists. Both TPO and IL-11, therefore, appear to be active thrombopoietic cytokines regulating, in part, megakaryocytopoiesis during states of acute thrombocytopenia.

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 beta-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.

No abstract