Rafanelli D

Inhibition of erythropoietin production in vitro by human interferon gamma.

The effects of interferon‐gamma (IFN‐γ). alone and in combination with IL‐1, IL‐6 and tumour necrosis factor‐alpha (TNF‐α), on in vitro erythropoietin (Epo) production by the human hepatoma Hep 3B cell line were evaluated. The addition of IFN‐γ to either unstimulated or cobalt chloride (CoCl2)‐treated Hep 3B cells resulted in a dosedependent inhibition of Epo release in the medium by as much as 70% at 1000 U/ml. Half‐maximal inhibition was observed at around 50 U/ml. According to previous observations, IL‐6 had a stimulatory effect on Epo production by CoCl2‐treated Hep3B cells: however, the simultaneous addition of IFN‐γ and IL‐6 resulted in a reversal of the stimulatory effects due to IL‐6. IFN‐γ and IL‐1 had an additive inhibitory effect, whereas IFN‐γ and TNF‐α acted in a synergistic fashion in inhibiting Epo production by Hep 3B cells. The inhibitory effect of IFN‐γ appeared to be due to a down‐modulation of Epo mRNA levels in CoCl2,‐treated Hep 3B cells, as shown by Northern blot analysis.

Recombinant human erythropoietin has little influence on megakaryocytopoiesis in mice

Summary. The availability of a preparation of recombinant human erythropoietin (rEp) prompted us to investigate the role of Ep in the regulation of megakaryocytopoiesis in mice, using experimental procedures by which the effects on the mitotic and post‐mitotic compartment of megakaryocytes could be evaluated separately. In agar cultures of murine bone marrow cells, either serum‐depleted or serum‐supplemented, rEp (0.1–2 U/ml) did not stimulate megakaryocyte colony formation and when it was added to suboptimal amount of spleen cell‐conditioned medium (SCM), it failed to show a significant synergistic activity. On the contrary, rEp increased the number of megakaryocytic colonies developed from splenic precursors in the presence of suboptimal amounts of SCM, although it was unable per se to stimulate colony formation.

No Hepatitis After Treatment with a Modified Factor IX Concentrate in Previously Untreated Hemophiliacs

Excerpt Post-transfusion hepatitis is a serious and frequent complication in patients treated with clotting factor concentrates made from plasma pooled from a large number of donors. The occurrence...

Protein content and factor VIII complex in untreated, treated and monoclonal factor VIII concentrates.

Replacement therapy with clotting factor concentrates may expose the recipients not only to virus contamination but also to continuous stimulation of the immune system by repeated infusions of allogenic proteins. Concentrate purity is now a very important prerequisite to be taken into account in choosing what product can better meet the patients needs. We compared protein content (albumin, fibrinogen, fibronectin, immunoglobulins) and factor VIII:C/vWF:Ag complex in untreated, treated and monoclonal factor VIII concentrates. Protein content is dramatically decreased in new treated ultrapure concentrates. Improved traditional fractionation methods allowed to obtain very high Factor VIII specific activity. New fractionation methods with immunoaffinity chromatography by means of monoclonal antibodies can give highly pure concentrates even if deliberately added albumin decreases factor VIII specific activity in final formulation. Otherwise monoclonal concentrates show a very high specific activity in terms of fibrinogen and immunoglobulin content, which, unlike albumin, are affecting the immune system in hemophiliacs.

Effects of miocamycin and erythromycin on polymorphonuclear cell function

Previous studies have shown that erythromycin can enter phagocytic cells, stimulating their functional activity. In this work we compared the effects of erythromycin and another newer macrolide antibiotic, miocamycin, on a series of in vitro tests aimed at evaluating their influence on polymorphonuclear cell (PMN) functions. Results indicate that erythromycin induces an increase in leukotriene B4 production in PMNs, while chemotaxis, killing of Candida albicans and respiratory burst are not influenced, at least at the doses used in this study. On the contrary, all these activities are significantly enhanced following incubation with miocamycin, and the response varies according to the antibiotic concentration.

A dot assay for the erythropoietin receptor using human recombinant 125I-erythropoietin

A dot assay was developed for the detection of membrane receptor(s) for erythropoietin (Ep). A relatively homogeneous population of cells bearing the receptor for Ep was generated in the spleen of mice made anemic with phenylhydrazine and crude membrane extracts were prepared from spleen cell suspensions. Aliquots of the membrane extracts were applied to microdishes of nitrocellulose in a volume of 4 microliters. After free reactive sites were blocked, the microdishes were incubated for 2 h at 37 degrees C with 125I-labeled human recombinant Ep (125I-rEp), and nitrocellulose bound radioactivity was determined thereafter. Reproducible curves were obtained, and a significant correlation between bound radioactivity and the amount of membrane proteins applied to the nitrocellulose dishes was found. Specific binding was saturable, reaching a plateau at 2.5 nM. Binding parameters of nitrocellulose-immobilized receptor were not significantly different from the values calculated using intact cells. No appreciable binding of 125I-rEp to control membranes at low Ep-receptor content was observed. Among a panel of growth factors, only unlabeled rEp was able to compete for the binding of 125I-rEp to nitrocellulose-immobilized membrane proteins in a dose-dependent fashion. The technique described herein may be of use in the study of the Ep receptor and as an assay for its purification. Moreover, it may also be of general application in the study of receptor-ligand interactions.

The Humoral Regulation of Normal and Pathologic Erythropoiesis

About 40 years ago it was recognized that a humoral factor, later identified as erythropoietin (Epo), was able to regulate the response of bone marrow to changes in red cell mass.1,2 It was also found that Epo was produced by the kidney in response to renal tissue hypoxia,3,4 and a quantitative assay based on the measurement of newly produced red cells labeled with59Fe was developed.2,5 The introduction of assays that allow cells to proliferate and differentiate in culture has led to the conclusion that Epo primarily acts on cells morphologically unrecognizable as erytroblasts. Semisolid cultures using methylcellulose, plasma-clot, and agar6–9 showed that two classes of erythroid progenitors can be observed at different times of incubation.7,8,10 Human bone marrow mononuclear cells in culture give rise to erythroid colonies after 4 to 5 days of incubation,11 while a longer (8–9 days) incubation time determines the appearance of larger colonies,7 and even larger and more hemoglobinized ones appear after 14 days. The first type is currently defined as colony-forming unit-erythroid (CFU-E)8; 14 and 8- to 9-day colonies are referred to as burst-forming unit-erythroid (BFU-E)12 and intermediate BFU-E,7 respectively (Fig. 3.1). Murine counterparts of these cells grow after 2 (CFU-E), 3 to 4 (intermediate BFU-E), and 8 (BFU-E) days.13 In addition, a murine progenitor with higher proliferative and differentiative capacity is seen after 9 to 12 days of incubation.7

Regulation of platelet production.

Our knowledge of the mechanisms regulating blood platelet production has received a boost in the last decade with the development of cloning assays in semi-solid and liquid media which permit the growth of megakaryocyte colony forming cells (CFUMk).lp4 The specificities of the methods that are used have also been improved by the introduction of specific cytochemical or immunological markers which recognize the megakaryocytic origin of the cells. Currently, acetylcholinesterase staining is used for murine colonies,’S6 and monoclonal antibodies directed against the platelet glycoproteins (GP Ib, IIb, IIIa, and IIb/IIIa complex) or platelet granule proteins (von Willebrand factor, vWf) are used to recognize human megakaryocyte colonies.