In vivo administration of stem cell factor enhances both proliferation and maturation of murine megakaryocytes.

BACKGROUND: Stem cell factor (SCF) has already been shown to participate in the regulation of erythro- and granulopoiesis. The aim of this study was to define the possible role of SCF in the regulation of megakaryocytopoiesis. METHODS: Stem cell factor activity has been assessed in an in vivo murine model, in which different doses of the factor were either given alone or in association with recombinant human erythropoietin (rhEpo). Mice were sacrificed after a six-day treatment to evaluate the effect of SCF on the number of bone marrow and spleen colony-forming units-megakaryocyte (CFU-Mk), and after a two-day treatment for evaluation of thrombopoietin-like activity. RESULTS: We found that SCF induces a dose-related increase in the number of CFU-Mk in both the bone marrow and spleen of the treated mice, and that in the range of the doses used (from 25 to 200 mg/kg/day) the greatest activity was observed when a dose of 200 mg/kg/day was injected. The effect was enhanced by adding rhEpo to optimal SCF concentrations. SCF also stimulated megakaryocyte maturation as assessed by the megakaryocyte number, the size of acetylcholinesterase-positive cells, 35Sulphur (35S) incorporation into the newly formed platelets. All these parameters were only minimally affected by the addition of rhEpo. CONCLUSIONS: These data suggest that SCF participates in the regulation of megakaryocytopoiesis and that its administration might have a role in the treatment of disorders of platelet production.

Early hemostatic alterations following bone marrow transplantation: a prospective study.

BACKGROUND: The occurrence of coagulation system alterations after bone marrow transplantation (BMT) and their possible role in the pathogenesis of thrombotic complications such as veno-occlusive disease of the liver (VOD) are still a matter of debate. The aim of this study was to evaluate prospectively the alterations in hemostatic balance developing during the early period after BMT (up to day +21) and their relationships (if any) with VOD. PATIENTS AND RESULTS: Twenty-nine patients (15 autologous and 14 allogeneic BMT) entered the study. No patient suffered from thrombotic and/or major hemorrhagic events. Since there were no differences between the two groups of patients with regard to modifications of coagulation parameters, they were considered together for the purposes of the study. We observed a progressive increase from baseline levels of fibrinogen, factor VIII activity (fVIII:C) and von Willebrand factor antigen (vWf), while factor VII antigen (fVIIAg), protein C and plasminogen significantly decreased. The alterations in these test values were maximal on day +14, with a trend towards normal levels one week later. There was no modification of PT, PTT, prothrombin fragment 1 + 2 (F 1 + 2), fXIIC, tPA, PAI-1, D dimer or protein S levels; serum levels of tumor necrosis factor-alpha were also unchanged. CONCLUSIONS: These results suggest that some alterations of the hemostatic system, probably a consequence of endothelial damage, can be detected early after BMT, but their clinical significance remains uncertain due to the lack of a correlation between hemostatic test alterations and the occurrence of thrombotic complications.

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.

Oral lesions among HIV-infected hemophiliacs. A study of 54 patients.

BACKGROUND: HIV-infected individuals develop a large variety of oral manifestations. This study was designed to assess the prevalence and types of oral lesions among HIV-positive hemophiliacs. MATERIALS AND METHODS: A study population of 54 hemophiliacs was evaluated from February, 1987 to March, 1992 in order to analyze types, prevalence and relationships to clinical stages of HIV-related oral lesions. Thirty-six (67%) of the group of patients were HIV seropositive. The remaining 18 tested negative to HIV during the observation period. RESULTS: The majority of patients suffered from hemophilia A. One patient was also bisexual and two were also intravenous drug abusers. Analysis of patient stage revealed that half had a CD4+ T-lymphocyte count over 0.5 x 10(9)/L cells, 10 between 0.2 and 0.499 x 10(9)/L and 8 showed a count lower than 200 x 10(9)/L. Oral lesions were recorded in 18 (50%) HIV-seropositive hemophiliacs. No oral lesions were observed among the HIV-seronegative hemophiliacs. Advanced stage of immunosuppression and presence of oral lesions were significantly associated (p = 0.040). Candidiasis was the most common disturbance, followed by hairy leukoplakia. Oral herpes simplex infection, necrotizing gingivitis and facial herpes zoster were found in a small number of patients. Those with oral lesions showed a lower median CD4+ T lymphocyte count (0.209 x 10(9)/L cells; range 0.008 to 0.615) when compared to the ones without oral lesions (median CD4+ count was 0.539 x 10(9)/L cells; range 0.042 to 1.180; p = 0.002). CONCLUSIONS: HIV-seropositive hemophiliacs may develop oral lesions during the course of their disease. Candidiasis and hairy leukoplakia are among the most common manifestations. A careful oral examination should be included in the clinical evaluation of all HIV-infected hemophiliacs.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Inadequate erythropoietin production in allogeneic bone marrow transplant patients.

BACKGROUND: There is some evidence that the erythropoietin (Epo) feedback mechanism in response to anemia can be altered in the period immediately following allogeneic bone marrow transplantation (BMT). METHODS: By using a RIA, serum erythropoietin (sEpo) levels were serially measured in 10 BMT patients, from day -10 up to day +30, and the results correlated with the concurrent hemoglobin (Hb) value. Thirty healthy subjects and 15 iron-deficiency anemic patients were used in order to construct our own reference sEpo vs Hb curve. A sEpo value recorded in BMT patients was considered to be inappropriate for any given Hb value when falling below the lower 95% confidence limit of the control curve. RESULTS: Basal sEpo levels were significantly higher than in healthy subjects, and increased further during the BMT procedure, being still higher than controls on day +30. However, Epo production resulted inappropriate for each given Hb value, when compared with the control curve, in 60 out of 67 sEpo determinations performed following graft infusion. The inadequate Epo production was not associated with the development of clinically manifest signs of kidney toxicity. CONCLUSION: These data indicate that Epo production is impaired in the period immediately following BMT and suggest a role for the administration of recombinant human Epo in the short-term management of the anemia associated with BMT.

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.

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.