R-h-erythropoietin counteracts the inhibition of in vitro erythropoiesis by tumour necrosis factor alpha in patients with rheumatoid arthritis

Anaemia of chronic disease (ACD) is a common extra-articular manifestation of rheumatoid arthritis (RA). Tumour necrosis factor alpha (TNFα) plays an important role in the development of ACD. The objective of the present study was to assess inhibition of in vitro colony-forming unit erythrocyte (CFUe) and blast-forming unit erythrocyte (BFUe) growth by TNFα and to examine whether this suppression could be counteracted by adding increasing concentrations of recombinant human erythropoietin (EPO) (r-h-EPO) to bone marrow cultures of RA patients with ACD and without anaemia (controls). Bone marrow cells of RA patients with ACD and control patients were cultured. The cultures were incubated with increasing concentrations of r-h-EPO (0.25; 0.5; 1; 2 U/ml), each in combination with increasing quantities of TFNα (0; 50; 100; 200; 400 U/ml). CFUe and BFUe were assessed after 7 and 14 days, respectively. Dose-dependent inhibition of BFUe and CFUc by increasing concentrations of TNFα was observed in ACD and controls. Regarding CFUe (ACD patients) incubated with 0.25 U/ml EPO, 50 U/ml TNFα caused 28% suppression compared to cultures without TNFα. Increasing the concentration of r-h-EPO from 0.25 U/ml to 2 U/ml completely restored the number of CFUe. A similar pattern was observed in BFUe growth in both groups. These data demonstrated the suppressive effects of TNFα on erythropoiesis in vitro and that the suppresed erythropoiesis could be partly corrected by the addition of excess r-h-EPO to the cultures. No significant differences were observed between ACD and control RA patients. This in vitro model may help explain the clinical response to r-h-EPO therapy as documented in RA patients with ACD

Interaction of inflammatory cytokines and erythropoeitin in iron metabolism and erythropoiesis in anaemia of chronic disease

In chronic inflammatory conditions increased endogenous release of specific cytokines (TNFα, IL-1, IL-6, IFNγ and others) is presumed. It has been shown that those of monocyte lineage play a key role in cytokine expression and synthesis. This may be associated with changes in iron metabolism and impaired erythropoiesis and may lead to development of anaemia in patients with rheumatoid arthritis. Firstly, increased synthesis of acute phase proteins, like ferritin, during chronic inflammation is proposed as the way by which the toxic effect of iron and thereby the synthesis of free oxy-radicals causing the damage on the affected joints, may be reduced. This is associated with a shift of iron towards the mononuclear phagocyte system which may participate in the development of anaemia of chronic disease. Secondly, an inhibitory action of inflammatory cytokines (TNFα, IL-1), on proliferation and differentiation of erythroid progenitors as well as on synthesis of erythropoietin has been shown, thereby also contributing to anaemia. Finally, chronic inflammation causes multiple, complex disturbances in the delicate physiologic equilibrium of interaction between cytokines and cells (erythroid progenitors, cells of mononuclear phagocyte system and erythropoietin producing cells) leading to development of anaemia of chronic disease (Fig. 1)

Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event