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Biomedical subjects

D Rafanelli

Publications and source records attributed to D Rafanelli.

At least 37 records · Page 2Linked to original sources
PubMed1989

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.

Anemia↗
PubMed1989

Recombinant human erythropoietin has little influence on megakaryocytopoiesis in mice.

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. The effects of rEps on megakaryocyte maturation and platelet production were studied in vivo evaluating the incorporation of 75Se-selenomethionine into platelets, the platelet count and platelet size, and the number of megakaryocyte precursors (small acetylcholinesterase positive cells, sAchE) in the bone marrow of mice injected with 1-8 U of rEp. No modification of these parameters was found in comparison with control mice. On the other hand, rEp increased the number of recognizable splenic megakaryocytes in a dose-dependent fashion. These data suggest that rEp has little influence on megakaryocytopoiesis, at least at the doses we used and which are known to elicit a maximal response of erythropoiesis. However, a subset of megakaryocytes with particular kinetic properties, such as those in the spleen of mice, may be responsive to relatively high doses of rEp. The significance of this observation in the overall regulation of megakaryocytopoiesis remains to be determined.

Animals↗
PubMed1988

Occurrence of haemorrhagic and thrombotic events in myeloproliferative disorders: a retrospective study of 108 patients.

Raised platelet count, impaired platelet function, and high PCV values are all considered risk factors for the haemorrhagic and/or thrombotic events frequently complicating the course of myeloproliferative disorders. Nevertheless, the outcome in young patients is reported as being frequently uncomplicated and, in these cases, treatment could be deferred. We examined retrospectively the occurrence of haemorrhagic and/or thrombotic events at the onset of myeloproliferative disorders in 108 patients and found that 67.4% of patients, with either raised platelet count or isolated high PCV, were symptomatic at the onset and the rate was similar regardless of disease, sex, age, platelet number and PCV values. We were unable to locate a patient group with a lower rate of complications and, in light of the frequency of symptoms, it seems reasonable to attempt a normalization of blood values in every case.

Adult↗
PubMed1988

Serotonin incorporation as a marker of murine megakaryocyte proliferation in liquid cultures.

We have developed a new in vitro method for the quantitation of murine megakaryocyte proliferation that is based on the unique property of megakaryocytes to incorporate and store [14C]serotonin in cytoplasmic dense granules. The specificity of the assay was demonstrated by autoradiography of whole bone marrow cell suspensions, which showed evidence of grain accumulation only in megakaryocytes. Bone marrow cells were cultured in liquid cultures in the presence of a stimulator of megakaryocyte growth before the addition of 2.5 microM [14C]serotonin. The amount of serotonin incorporated in cells was evaluated after 3 h. Radioactivity peaked at days 6 and 7 and remained high until day 10; there was a linear relationship between the incorporation of serotonin and the number of cells plated. A dose-response curve between the incorporation of serotonin and the concentration of pokeweed mitogen spleen-conditioned medium (PWM-SCM) was observed, with inhibitory effects becoming predominant at the highest concentrations. The proliferation of megakaryocyte progenitors was also stimulated by partially purified interleukin 3, whereas both human recombinant erythropoietin and human recombinant granulocyte colony-stimulating factor (rG-CSF) failed to modify the incorporation of serotonin in comparison with unstimulated cultures. Finally, in parallel experiments we observed a significant correlation between the number of megakaryocytic colonies grown in agar and the radioactivity in liquid cultures. The method described herein is reproducible, sensitive, and easy to perform; it should be useful for the study and purification of factors affecting megakaryocyte proliferation.

Animals↗
PubMed1988

Partial purification and biochemical characterization of human plasma thrombopoietin.

A factor that stimulates the incorporation of 75Selenomethionine into the newly formed platelets of recipient mice (thrombopoietin, TPO) has been partially purified from the plasma of thrombocytopenic patients. The activity was precipitated at 60-80% ammonium sulfate saturation and further purified with hydrophobic interaction chromatography. Thrombopoietin was retained by concanavalin-A-Sepharose. Using HPLC size-exclusion chromatography, an approximate molecular weight of 40,000 dalton was calculated. The overall purification factor was about 2,100-fold. TPO was stable in a pH range from 5 to 9 and was heat-sensitive, and the biological activity was destroyed by trypsin treatment and by dithiothreitol. The partially purified molecule did not stimulate the proliferation of megakaryocyte progenitors in vitro and had no effect on the growth of erythroid or granulocyte-macrophage colonies; when administered in-vivo, TPO significantly affected the mean platelet volume and increased the number of small acetylcholinesterase cells in the bone marrow. TPO appears to be specific for the megakaryocytic lineage and active on the postmitotic compartment of megakaryocytes.

Glycoproteins↗
PubMed1987

Megakaryocyte progenitors in the bone marrow and peripheral blood of patients with myeloproliferative diseases.

We studied the behavior in culture of megakaryocyte progenitor cells (CFU-Mk) from peripheral blood (PB) and bone marrow (BM) cells in eight patients with myeloproliferative diseases (MPD). In seven patients we observed megakaryocyte (Mk) colony formation from PB cells, which were generated in the absence of any added stimulator and which did not increase after the addition of a source of Mk-colony stimulating activity (CSA-Mk). The number of BM CFU-Mk was significantly higher in patients than in controls, and in seven out of eight patients the responsiveness to added CSA-Mk was retained. Plasma obtained from six patients did not stimulate normal donors' BM target cells to form Mk colonies. These data demonstrate an expansion of the CFU-Mk pool in MPD patients without increased plasma levels of CSA-Mk, and suggest that PB and BM CFU-Mk of MPD patients might have different kinetic properties.

Bone Marrow Cells↗
PubMed1986

Hepatitis-free interval after clotting factor therapy in first infused haemophiliacs.

Post-infusion hepatitis is known to occur very frequently in haemophiliacs after treatment with unheated commercial clotting factor concentrates, obtained from large plasma donation pool. On the contrary, single-donor cryoprecipitate is likely to carry a lower risk of transmitting hepatitis. To evaluate this hypothesis, we retrospectively reviewed the medical records of 25 first infused haemophiliacs (from 1981 to 1984) treated with unheated commercial clotting factor concentrates (n = 19) or cryoprecipitate (n = 6). The hepatitis-free interval after the beginning of therapy was expressed as exposure days. The end point of each patient, i.e. the hepatitis occurrence, was defined as an increase of amino-transferases (ALT and AST) and/or the seroconversion of HBV-markers, which were checked every three months. The life-table method and log-rank test showed that cryoprecipitates had a significantly longer hepatitis-free interval (p = 0.0131, log-rank test) and a lower risk of transmitting hepatitis (p = 0.01-0.05, life-table method) than the commercial concentrates. However, the safety of cryoprecipitate therapy was shown to cover only a few exposure days, and so the real advantage of this product depends on the bleeding frequency of the patient concerned. We believe that these methods and our findings may be useful to assess and compare the safety of the new "heat-treated" clotting factor concentrates.

Adolescent↗
PubMed1986

Platelet function and coagulation studies in patients with mitral valve prolapse.

Twenty-eight consecutive patients with mitral valve prolapse (MVP), seven of whom had previous cerebrovascular disorders (CVD), were studied for platelet function and coagulation tests. While platelet function tests were found to be normal with the exception of platelet aggregation rate (PAR), there was a significant rise of factors VIII vWF:Ag (Von Willebrand) and (FPA) fibrinopeptide A. Six cases had high levels of both these factors, suggesting the existence of a particular subset of patients with MVP, with a higher risk of thromboembolic episodes, although only three out of seven patients with previous CVD had either FPA or VIII vWF:Ag levels. The broad spectrum of subjects with MVP probably explains the different results obtained when studying platelet function and coagulation factors. Therefore, larger population studies and prolonged follow-up of cases with either coagulation abnormalities similar to the ones found in the present report and/or altered platelet function tests are suggested to discover if it is possible to detect patients with a potential for thromboembolism.

Adolescent↗
PubMed1986

Pharmacological activity of FPP028 (2-phenylpyrazolo-4-ethyl-4,7-dihydro [1,5a]pyrimidin-7-one) a new non-steroid anti-inflammatory agent.

The activity of 2-phenylpyrazolo-4-ethyl-4,7-dihydro [1,5a]pyrimidin-7-one (FPP028), a non-acidic, analgesic, antipyretic, and anti-inflammatory compound, was investigated in a number of pharmacological tests performed in rats. The anti-inflammatory properties of FPP028 were evaluated through the carrageenan induced paw edema and the cotton pellet induced granuloma and compared with the activity of indomethacin, phenylbutazone, and isoxicam; as a result, the activity of FPP028 was shown to be similar to that of the latter compounds. To assess the analgesic properties of FPP028 in comparison with indomethacin and phenylbutazone, the Randall and Sellitto and the mouse-writhing tests were used; in both tests, FPP028 demonstrated a significant analgesic activity. FPP028 was shown to possess antipyretic properties in the test of yeast-induced pyrexia. The gastro-erosive activity of phenylbutazone and FPP028 was studied in restraint-stressed rats; in such test the ulcerogenic activity of phenylbutazone appeared to be dose-related; conversely, FPP028 demonstrated a gastro-protective effect since the number of gastric lesions induced either by stress or phenylbutazone treatment was decreased bu FPP028. Our data show that FPP028 is endowed with most of the pharmacological properties of the classic antiinflammatory drugs. Further studies are however needed to more fully elucidate its mechanism of action because our in-vivo data indicate that FPP028 is not an inhibitor of prostaglandin biosynthesis.

Adrenalectomy↗
PubMed1986

Abnormalities of in vitro immunoglobulin production in apparently healthy haemophiliacs: relationship with alterations of T cell subsets and with HTLV-III seropositivity.

The pokeweed mitogen (PWM)-induced immunoglobulin (Ig) production by cultures of peripheral blood mononuclear cells (PBMC) was reduced in healthy haemophiliacs treated with commercial factor VIII (or IX) concentrate, whereas the spontaneous IgG synthesis in vitro was enhanced. PWM-induced Ig production was lower in those who had received greater amounts of concentrate, in those with inverted T4/T8 lymphocyte ratios and in those with antibody to HTLV-III. The spontaneous IgG production in vitro was higher in haemophiliacs who had received larger amounts of concentrate, in those with inverted T4/T8 ratio and in those with antibody anti-HTLV-III. However, some patients with normal T4/T8 ratio and some with HTLV-III antibody also had raised spontaneous IgG production.

Antibodies, Viral↗
PubMed1983

Different patterns of platelet turnover in chronic idiopathic thrombocytopenic purpura.

Platelet turnover, platelet production, platelet mean life span (MLS), platelet count, mean platelet volume (MPV) and platelet-associated antibodies have been examined in 26 patients with chronic idiopathic thrombocytopenic purpura (ITP) and in 1 patient with hypomegakaryocytic thrombocytopenia (HT). 15 ITP patients had normal or increased platelet turnover and platelet production, while 11 had subnormal values despite shortened MLS, while the patient with HT had normal MLS. The differences between the two groups with high and low platelet turnover were statistically significant. No correlation was found between kinetics parameters and bone marrow pattern in a total of 19 patients examined. These data suggest that in some cases of chronic ITP, the pathogenesis of thrombocytopenia can be due not only to the peripheral destruction of platelets, but also to a deficient platelet production by megakaryocytes. Since the number of megakaryocytes in bone marrow slides is not decreased in the low turnover compared with the high turnover group, it is possible that an impaired pattern of megakaryocyte maturation be the cause of the low platelet production in these patients, unlike in the HT patient where megakaryocytes are almost absent.

Adolescent↗