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D Rafanelli

Publications and source records attributed to D Rafanelli.

At least 19 recordsLinked to original sources
PubMed1994

Inhibition of erythropoietin production in vitro by human interferon gamma.

The effects of interferon-gamma (IFN-gamma), alone and in combination with IL-1, IL-6 and tumour necrosis factor-alpha (TNF-alpha), on in vitro erythropoietin (Epo) production by the human hepatoma Hep3B cell line were evaluated. The addition of IFN-gamma to either unstimulated or cobalt chloride (CoCl2)-treated Hep3B cells resulted in a dose-dependent 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-gamma and IL-6 resulted in a reversal of the stimulatory effects due to IL-6. IFN-gamma and IL-1 had an additive inhibitory effect, whereas IFN-gamma and TNF-alpha acted in a synergistic fashion in inhibiting Epo production by Hep3B cells. The inhibitory effect of IFN-gamma appeared to be due to a down-modulation of Epo mRNA levels in CoCl2-treated Hep3B cells, as shown by Northern blot analysis. These data indicate that Epo production by hepatoma cells in vitro is inhibited by IFN-gamma, and that a complex network of interacting cytokines may regulate Epo production in response to an hypoxic stimulus. Overall, these results also suggest that IFN-gamma might have a role in the defective Epo production observed in several inflammatory and immunemediated disorders characterized by relatively high IFN-gamma plasma levels.

Blotting, Northern↗
PubMed1994

Prevalence of infection with the hepatitis C virus among Italian hemophiliacs before and after the introduction of virally inactivated clotting factor concentrates: a retrospective evaluation.

In July 1985, all coagulation factor concentrates were withdrawn from the market in Italy and replaced with virally inactivated concentrates. A retrospective survey comparing the prevalence of the antibody to the hepatitis C virus (anti-HCV) in hemophiliacs multitransfused with nonvirally inactivated concentrates until 1985 with that in previously untreated hemophiliacs transfused exclusively with virally inactivated concentrates since 1985 has been conducted in 9 Italian hemophilia centers. The centers, which follow about one-fourth of all the Italian hemophiliacs, provided information about 708 patients infused for the first time before 1985 (group A) and 80 patients infused for the first time between 1985 and 1991 (group B). The prevalence of anti-HCV was 83% (591/708) in group A and 6% (5/80) in group B. For the 5 anti-HCV-seropositive patients from group B, dry heating, hydrophobic interaction chromatography plus dry heating (2 patients), hot vapor and pasteurization were the virucidal methods used for the concentrates implicated in HCV transmission. In the case associated with pasteurization, there is the possibility of intrafamilial transmission of HCV. It appears from this retrospective analysis that there has been a substantial reduction in the risk of HCV transmission since the adoption of virucidal methods. However, these methods do not eliminate completely the risk, which might be further reduced by the recent adoption of anti-HCV screening for plasma donations used to manufacture concentrates.

Blood Coagulation Factors↗
PubMed1993

Effects of cyclosporin A on erythropoietin production by the human Hep3B hepatoma cell line.

There is evidence that the inadequate erythropoietin (Epo) production observed in patients undergoing allogeneic bone marrow transplantation (BMT) might be ascribed to an inhibitory effect caused by the immunosuppressive drug cyclosporin A (CsA). In this in vitro study, we have evaluated the effects of CsA on the release of Epo in the culture medium by the human Hep3B hepatoma cell line. In cultures incubated with both CsA and the nonimmunosuppressive CsA analog MeAla-6, but not with the CsA-unrelated immunosuppressive agent FK-506, the levels of Epo in the medium were significantly reduced in comparison with controls, at concentrations (0.01 to 1.6 mumol/L) not affecting total protein synthetic rate nor the constitutive secretion of alpha-fetoprotein. Hep3B cells were found to contain a CsA-binding molecule, with an M(r) of 18 Kd, as assessed by high performance liquid chromatography (HPLC) and ligand-blotting analysis. CsA did not affect the expression of the Epo gene, as judged by Northern blot analysis, but caused a significant amount of Epo to remain unsecreted within the cells; almost all (97% of total) of the intracellular Epo was associated with the plasma membrane subcellular fraction. We conclude that: (1) CsA is able to inhibit Epo release in vitro by Hep3B cells, further supporting the hypothesis that the drug might have a role in the inappropriately low Epo levels observed in BMT patients; (2) the inhibitory effect appears to be specific and not caused by a general impairment of protein synthesis and/or secretion; and (3) the reduced Epo levels found in the medium of CsA-treated Hep3B cultures are supposed to be the consequence of an inability of the cells to correctly process Epo molecules for the secretory pathway.

Amino Acid Isomerases↗
PubMed1993

Accelerated schedule of hepatitis B vaccination in patients with hemophilia.

Early development of immunity after hepatitis B vaccination is particularly important for patients such as hemophiliacs, at high risk for acquiring hepatitis B from potentially infectious plasma-derived concentrates. The purpose of this study was to evaluate whether or not protective antibody titers could be achieved quickly and maintained in hemophiliacs by an accelerated vaccination schedule. A yeast-recombinant hepatitis B vaccine (Engerix B, SKF Ritt) was given subcutaneously in the deltoid region and repeated 2 and 6 weeks later to 85 hemophiliacs negative for hepatitis B virus (HBV) markers. After the first 22 patients had been enrolled, a modification of the schedule involving a fourth booster dose 24 weeks after the first dose of vaccine was applied to the next 63 consecutive vaccines. Fifty-three percent of vaccinees had antibody titers to hepatitis B surface antigen (anti-HBs > or = 10 mlU/ml) by week 6, even though the mean titers of anti-HBs were somewhat lower than those achieved historically in normal individuals. The protection rate had increased to 87% by week 10, one month after the third dose of vaccine, and to 93% by week 24. One year after starting vaccination, the rate for the vaccinees who did not receive the fourth booster dose was 71%, and 96% for those who did receive the fourth dose, with only 2 patients not responding despite the booster dose. It is concluded that even though the accelerated schedule of immunization produced rapidly high rates of protective antibody titers, a booster dose is required to obtain higher titers and provide more persistent immunity.

Adolescent↗
PubMed1993

Prognostic value of a novel circulating serum 90K antigen in HIV-infected haemophilia patients.

We evaluated the prognostic role of a novel tumour-associated antigen, termed 90K, in a cohort of HIV+ asymptomatic haemophilia patients with known duration of seropositivity and median follow-up of about 7 years. The circulating levels of 90K are higher in HIV+ asymptomatic patients than HIV- controls. The antigen levels remain quite stable over time in non-progressing patients, while they steadily rise in patients evolving to ARC/AIDS. Baseline high 90K levels are predictive of faster progression to ARC/AIDS and shorter survival. We conclude that an elevated 90K serum level is a predictor of poor prognosis in HIV+ asymptomatic haemophiliacs.

AIDS-Related Complex↗
PubMed1992

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.

Candida albicans↗
PubMed1992

Human parvovirus B19 in clotting factor concentrates: B19 DNA detection by the nested polymerase chain reaction.

The presence of B19 parvovirus in plasma from blood donors is seldom demonstrable, but clotting factor concentrates, prepared from large plasma pools, may be able to transmit B19 virus infection, and the effectiveness of different chemical and physical treatment to inactivate this virus is not yet known. In this study we report on the detection of B19 DNA in 25 clotting factor concentrates, prepared by a variety of procedures of purification and inactivation; dot blot hybridization and Southern blot hybridization assays, as well as a 'nested' polymerase chain reaction (PCR) have been employed. Nine out of 25 products were B19 DNA positive by PCR, whereas only two gave positive results by hybridization techniques. B19 DNA positive concentrates have been found in 'untreated' products but also in some solvent/detergent or steam-treated products and even in monoclonal purified concentrates. PCR may be useful for the screening of blood products to be used in immunocompromised haemophiliacs, particularly in HIV positive subjects, at risk of severe chronic anaemia following B19 infection.

Base Sequence↗
PubMed1992

Constitutive expression of a megakaryocytic functional property by murine erythroleukemia (Friend) cells: the incorporation of serotonin.

Murine Friend-derived erythroleukemia cells (MEL) are generally believed to be unipotential progenitors inducible to terminal erythroid differentiation. However, we found that MEL can constitutively incorporate significant amounts of radiolabeled serotonin ([3H]5-HT). Because this process is typical of cells belonging to the megakaryocytic lineage, we investigated the significance and mechanisms of 5-HT incorporation in the MEL system. We observed that: 1) normal murine erythroid cells and erythroid progenitors do not incorporate [3H]5-HT, as well as normal murine myeloid cells and the human myeloid cell line HL-60; on the other hand, the human erythroleukemia cell lines K562 and HEL, which have been shown to constitutively express megakaryocytic features, were able to incorporate [3H]5-HT; 2) MEL incorporated 5-HT by an active and saturable mechanism, dependent on temperature and sodium concentration in the medium; and 3) 5-HT uptake was very rapid. Moreover, because about 65% of cell-associated radioactivity was no longer displaced by the cold substrate, we assumed it to represent "true" cytoplasmic internalization. Finally, 5-HT incorporation by MEL was inhibited by clomipramine, ouabain, and reserpine, which are known inhibitors of 5-HT uptake in platelets. The commitment of MEL to terminal erythroid differentiation by hexamethylene bisacetamide or dimethyl sulfoxide greatly reduced the capacity to incorporate [3H]5-HT. These results seem to suggest that the MEL system, although mainly erythroid as regards its differentiation capability, constitutively expresses features of the megakaryocytic lineage, possibly disclosed by the ability to incorporate 5-HT. This hypothesis was further supported by the findings that 30%-40% of uninduced MEL were labeled by a polyclonal antibody raised against murine platelets that selectively recognized megakaryocytes in murine bone marrow smears.

Acetamides↗
PubMed1992

Recombinant human erythropoietin for treatment of myelodysplastic syndromes.

Nine patients with myelodysplastic syndromes and one patient with agnogenic myeloid metaplasia have been treated with recombinant human erythropoietin (rhEpo), at the dose of 150 U/kg/day. Although serum Epo levels were correlated with hemoglobin concentrations in the whole population of patients, they clearly appeared inadequate in some instances, if compared to those of a group of control subjects with iron deficiency anemia. Moreover, no correlation was found between serum Epo and reticulocytes. Six patients showed a partial or complete response to the treatment and the outcome was not correlated with the pre-therapy serum Epo levels; however, serum Epo was less than 100 mU/ml in three of four patients who achieved a complete response. The mechanism(s) by which Epo stimulated erythrocyte production in myelodysplastic patients is unclear, because the number of both the reticulocytes and erythroid progenitors remained unchanged during and at the conclusion of a three months' therapy. Further studies are needed to better define the optimal dosage required to correct anemia in myelodysplastic syndromes, and to clarify rhEpo mechanism of action in these diseases.

Aged↗
PubMed1991

Impaired erythropoietin production in mice treated with cyclosporin A.

Because recent data indicate that erythropoietin (Epo) production is defective in allogeneic bone marrow transplant (BMT) patients, we investigated the role of the immunosuppressant, nephrotoxic, agent cyclosporin A (CsA) on renal Epo production using an animal model. Mice were injected with 1.0 to 40.0 mg/kg/d CsA for 15 days. Thereafter, circulating Epo levels were evaluated in both intact animals and in mice made anemic with phenylhydrazine (PHZ). Serum Epo levels measured in CsA-treated animals were then compared with the predicted levels, which had been calculated in a reference population of normal, either intact or anemic, mice. In CsA-treated, intact animals both hematocrit and serum Epo levels were not significantly different from controls. However, serum Epo levels in CsA-treated, anemic mice were significantly lower than those expected in a control population of untreated, anemic mice with similar degrees of anemia. No significant increase in serum creatinine was recorded even at the highest doses of CsA used, nor were we able to document signs of renal toxicity by histologic examination of the kidneys. Therefore, therapeutical doses of CsA appear to affect the production of Epo under conditions in which the demand of the hormone is increased, as in response to anemia. We suggest that a subclinical kidney toxicity produced by CsA might have a role in the pathogenesis of the impaired Epo production observed in BMT patients, and may contribute to a delayed erythroid engraftment in at least some BMT patients.

Anemia↗
PubMed1991

Serum erythropoietin levels in patients undergoing autologous bone marrow transplantation.

Serum erythropoietin (sEpo) levels were serially measured with a radioimmunoassay in 14 patients undergoing autologous bone marrow transplantation (BMT), starting before the institution of the conditioning regimen up to day +45. An increase in sEpo levels was observed soon after starting the chemotherapy regimen, and before an evident fall in hemoglobin (Hb) levels took place. The peak in sEPo levels (221 +/- 181 mU/ml) was reached at day 0 in 9/14 patients, and was delayed up to day + 10 in the remaining five. There was a negative correlation between loge sEpo and Hb values (r = -0.730; p less than 0.01); the regression line of this correlation was comparable to the one obtained in a group of 15 iron-deficiency anemic subjects. Therefore, patients undergoing autologous BMT appear to be able to develop adequately increased sEpo levels in response to the severity of anemia. No correlation was found between sEpo and white blood cell or platelet count. On the other hand, sEpo value at day 0 was significantly related to the day of neutrophil recovery (r = -0.806; p less than 0.001): patients with the highest sEpo levels at day 0 showed significantly faster (p less than 0.001) neutrophil recovery.

Adolescent↗
PubMed1990

In vivo stimulation of megakaryocytopoiesis by recombinant murine granulocyte-macrophage colony-stimulating factor.

Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was injected in mice, and the effects on bone marrow, splenic megakaryocytes, megakaryocyte precursors (megakaryocyte colony-forming units [CFU-Meg]) were evaluated. In mice injected three times a day for 6 days with 12,000 to 120,000 U rGM-CSF, no significant modification of both platelet levels and mean platelet volume was observed, while there was a twofold increase in blood neutrophils. However, the rate of platelet production, as assessed by the measurement of 75selenomethionine incorporation into blood platelets, was On the contrary, administration of up to 384,000 U rGM-CSF two times a day for 2 days, as for a typical "thrombopoietin assay," failed to modify platelet production. A significant dose-related increase in the number of splenic megakaryocytes occurred in mice receiving 60,000 to 120,000 U rGM-CSF, while a slight increase in the number of bone marrow megakaryocytes was observed in mice injected with 120,000 U rGM-CSF. The proportion of bone marrow megakaryocytes with a size less than 18 microns and greater than 35 microns resulted significantly higher in mice receiving rGM-CSF in comparison with controls; an increase in the percentage of splenic megakaryocytes greater than 35 microns was also observed. A statistically significant increase in the total spleen content of CFU-Meg was observed after administration of 90,000 and 120,000 U rGM-CSF three times a day for 6 days, while no effect on bone marrow CFU-Meg was recorded, irrespective of the dose delivered. Finally, 24 hours after a single intravenous injection of rGM-CSF, there was a significant increase in the proportion of CFU-Meg in S-phase, with the splenic progenitors being more sensitive than bone marrow-derived CFU-Meg. These data indicate that rGM-CSF has in vivo megakaryocyte stimulatory activity, and are consistent with previous in vitro observations. However, an effective stimulation of megakaryocytopoiesis in vivo, bringing about an increase in the levels of blood platelets, may require interaction of rGM-CSF with other cytokines.

Animals↗
PubMed1989

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 patient's 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.

Antibodies, Monoclonal↗
PubMed1989

Does ergometric stress test induce a procoagulative condition in patients with previous myocardial infarction?

A regularly scheduled physical training program seems to have antithrombotic effects. Moreover, the hemostatic changes occurring in patients with coronary artery disease during acute exercise have not been clearly elucidated. Since stress testing is routinely performed in clinical cardiology, it would be helpful to assess whether patients with coronary artery disease are exposed to acute coronary thrombosis during or soon after sustained physical exercise. This study was designed to evaluate the effect of acute physical exercise (stress test by bicycle ergometer) on blood coagulation in a group of patients with previous myocardial infarction, and to determine whether the antithrombotic therapy commonly administered favorably influences hemostatic equilibrium. Our results suggest that exercise testing is not harmful to patients with previous myocardial infarction in regard to hemostasis and fibrinolysis and that antithrombotic therapy reduces postexercise increase in platelets.

Aged↗