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To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P

Objective: To investigate telomere length (TL) and hematopoietic progenitors in long-term survivors after high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft., Methods: Peripheral blood (PB) and bone marrow (BM) samples were obtained from 31 subjects in continuous complete remission from a high-risk lymphoma, at a median of 5.8 years (range: 1-11 years) since autograft. Most of them were grafted with large PBSC quantities (median CD34(+ve) cells/kg: 7 x 10(6)). TL was determined by Southern blot analysis, BM progenitors by in vitro long-term culture-initiating cells (LTC-IC) and colony assays., Results: TL of PB granulocytes was significantly shortened in autografted subjects compared with age-matched healthy subjects; a similar finding was observed in BM. The median TL reduction in granulocytes from autografted subjects compared with age-matched controls (Delta(TelShortening)) was then assessed according to time interval since autograft. Three subject subgroups were identified-at 1 to <3 years, 3 to <6 years, and 6 to 11 years since autograft-and their telomere loss was the same, with Delta(TelShortening) of 1132, 1379, and 1214 bp in the three subgroups, respectively. The longitudinal assessment of TL in five representative patients followed for up to 40 months since autograft confirmed that telomere shortening occurring during exposure to chemotherapy as well as postautograft is persistent at long term. BM LTC-IC and multipotent and committed progenitors were assessed in subjects at >3 years after autograft and found to be markedly reduced compared with normal controls., Conclusion: High-dose chemotherapy and PBSC autograft may result in myelopoietic cell abnormalities that appear to be irreversible.

The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs), high-proliferative potential-CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). In mice, both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs, whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold, respectively, as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone, suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys, rhPlGF-1 alone had no mobilization effect, whereas rhPlGF-1 (260 microg/kg per day) plus rhG-CSF (100 microg/kg per day) increased rhG-CSF-elicited mobilization of CFCs, HPP-CFCs, and LTC-ICs per milliliter of blood by 5-, 7-, and 15-fold, respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion, our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting.

Objective: To explore new treatments specifically targeting malignant plasma cells (PCs), we examined CD52 antigen expression on primary PCs as well as multiple myeloma (MM) cell lines, and investigated in vivo the antimyeloma activity of alemtuzumab., Materials and Methods: PCs were enriched from the marrow of MM patients (n = 39) according to CD138 expression and then analyzed by 3-color flow cytometry and quantitative PCR. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice., Results: CD52 expression revealed a substantial heterogeneity in terms of both percentage of positive cells and fluorescence intensity, with 25/39 (64%) MM patients showing >or=30% CD138(+) PCs expressing the CD52 antigen (mean = 79%; range, 33-100%). Similarly to primary cells, cell lines showed heterogeneous CD52 expression. Expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. In vivo, alemtuzumab treatment significantly increased the median survival of animals with an early- (64 vs 77 days, p

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.

Objectives: To assess the pharmacological treatment and the control of major modifiable cardiovascular risk factors in everyday practice according to the patients' cardiovascular risk level., Methods: In a cross-sectional study general practitioners (GPs) had to identify a random sample of their patients with cardiovascular risk factors or diseases and collect essential data on the pharmacological treatment and control of hypertension, hyperlipidemia, and diabetes according to the patients' cardiovascular risk level and history of cardiovascular disease. Participants were subjects of both sexes, aged 40-80 years, with at least one known cardiovascular risk factor or a history of cardiovascular diseases., Results: From June to December 2000, 162 Italian GPs enrolled 3120 of their patients (2470 hypertensives, 1373 hyperlipidemics, and 604 diabetics). Despite the positive association between the perceived level of global cardiovascular risk and lipid-lowering drug prescriptions in hyperlipidemic subjects (from 26% for lowest risk to 56% for highest risk p < 0.0001) or the prescription of combination therapy in hypertensives (from 41% to 70%, p < 0.0001) and diabetics (from 24% to 43%, p = 0.057), control was still inadequate in 48% of diabetics, 77% of hypertensives, and 85% of hyperlipidemics, with no increase in patients at highest risk. Trends for treatment and control were similar in patients with cardiovascular diseases., Conclusions: Even in high-risk patients, despite a tendency towards more intensive treatment, pharmacological therapy is still under used and the degree of control of blood pressure, cholesterol level and diabetes is largely unsatisfactory.

Objective: In vivo administration of alemtuzumab (an anti-CD52 antibody) is effective to decrease the incidence of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). However, posttransplant immune reconstitution is impaired, increasing the infection risk. We investigated the effect of in vivo administration of a low-dose alemtuzumab on GVHD prevention and kinetics of immune reconstitution., Patients and Methods: Twenty-seven patients entered a pilot study employing reduced-intensity conditioning and low-dose alemtuzumab (15 or 7.5 mg/m2) before peripheral blood allo-SCT from human leukocyte antigen-identical or one antigen-mismatched sibling donors. All lymphoid subsets were longitudinally studied at 1-3, 6, 9, 12 months after transplantation. T-cell receptor (TCR) spectratyping and T-cell receptor excision circles (TRECs) were also analyzed at various time points after allo-SCT., Results: All patients but one were engrafted. The probability of nonrelapse mortality at 100 days and 1 year were 7 and 11%, respectively; the overall survival at 2 years was 77%. The cumulative incidence of grade II-IV acute GVHD at day 100 was 11%. The overall incidence of chronic GVHD was 28%. The median time to achieve more than 200 CD4+/microL and 500 CD8+/microL were 6 and 9 months, respectively. Natural killer cells remained between the value of 300/microL and 500/microL throughout the period of follow-up whereas the median time to reach CD19+ blood concentrations of >200 cells/microL was 9 months. The normalization of TCR repertoire and increase of TREC counts began at 6 months after allo-SCT., Conclusion: We have shown that low-dose alemtuzumab is effective for GVHD prevention, but its use still impairs the immune reconstitution.

Background: Although risk assessment charts have been proposed to identify patients at high cardiovascular risk, in everyday practice general practitioners (GPs) often use their knowledge of the patients to estimate the risk subjectively., Design: A cross-sectional study aimed to describe how GPs perceive, qualify and grade cardiovascular risk in everyday practice., Methods: General practitioners had to identify in a random sample of 10% of their contacts the first 20 consecutive patients perceived as being at cardiovascular risk. For each patient essential data were collected on clinical history, physical examination and laboratory tests, for the qualification of risk. At the end of the process GPs subjectively estimated the overall patient's level of risk. General practitioners grading was compared with the risk estimate from a reference chart., Results: Over a mean time of 25 days 3120 patients perceived as being at cardiovascular risk were enrolled. According to the inclusion scheme each GP had contact with more than 200 patients at cardiovascular risk every month. Thirty percent of these patients had atherosclerotic diseases. Up to 72% of patients without any history of atherosclerotic diseases but perceived to be at risk could be classified according to a reference chart as being at moderate to very high risk. Comparing GPs' grading of risk with a chart estimate there was agreement in 42% of the cases. Major determinants of GPs' underestimation of risk were age, sex and smoking habits, while obesity and family history were independently associated with overestimation., Conclusions: On the basis of their perception GPs properly identify patients at cardiovascular risk in the majority of cases. General practitioners subjective grading of risk level only partially agreed with that given by a chart.

Objective: The aim of this story was to evaluate the activity of recombinant human (rh) growth hormone (GH) in restoring bone marrow progenitor cell growth as well as cytokine-elicited stem cell mobilization in aged BALB/c mice with impaired marrow hematopoietic function and reduced stem cell mobilizing capacity., Materials and Methods: BALB/c mice included in this study were either naturally aged (group I) or aged after having been used for radioprotective assays (group II). Mice were treated for 5 weeks with either rhGH [2.5 mg/kg/day intraperitoneally (IP)] or phosphate-buffered saline (PBS). Subsequently, colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) were evaluated. In addition, progenitor cell mobilization elicited by granulocyte colony-stimulating factor (rhG-CSF) was analyzed., Results: Compared with young controls, the growth of marrow CFCs and LTC-ICs was significantly reduced (P < or = 0.05) in group I and II mice. Treatment with rhGH significantly enhanced marrow hematopoiesis in mice of both groups, as demonstrated by a complete restoration of marrow cellularity, and CFC and LTC-IC growth. To further evaluate the hematopoietic potential of rhGH, aged mice treated with rhGH or PBS were mobilized with rhG-CSF (10 microg/day IP for 5 days). Compared with PBS-injected mice, rhGH-treated mice showed a significant improvement of rhG/CSF-elicited stem cell mobilization, with significant increases of white blood cell counts (5633 vs 8133, P < or = 0.05), frequency of circulating CFCs per 10(5) mononuclear cells (36 vs 67, P < or = 0.009), as well as absolute numbers per mL of blood of circulating CFCs (783 vs 2288, P < or = 0.001) and LTC-IC (21 vs 64, P < or = 0.001)., Conclusion: Our data demonstrate in mice that a 5-week treatment with rhGH restores age- and irradiation-associated loss of marrow primitive and committed progenitors.

Objective: The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys., Materials and Methods: Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 microg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs)., Results: Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, p< or =0.02), 6- (4358 vs 748, p< or =0.02), and 5-fold (884 vs 168, p< or =0.04), respectively. We then evaluated the effects of a 2-day defibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, p< or =0.05), 8- (5361 vs 660, p< or =0.01), and 8-fold (954 vs 119, p< or =0.01), respectively., Conclusion: Our data demonstrate that in nonhuman primates: 1) defibrotide enhances rhG-CSF-elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P < or = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < or = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (P < or = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < or = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.

We have evaluated, as a vector for gene transfer into human T lymphocytes, a recombinant adenovirus (rAd-MFG-AP) carrying a modified, membrane-exposed, alkaline phosphatase (AP) as reporter gene. CD3+ cells were selected from the buffy coat of healthy donors by the immunomagnetic technique. The positive cell population, comprising 96+/-2% CD3+ cells, was cultured with clinical-grade cytokine(s) for 3-7 days prior to rAd-MFG-AP transduction and the transgene expression was evaluated 48 hr later by indirect immunofluorescence flow cytometry assay with an anti-alkaline phosphatase antibody. The best efficiency of transduction was achieved on incubation of CD3+ cells with IL-2 plus either IL-12 (AP+ cells, 12+/-3%) or IL-7 (AP+ cells, 11+/-3%). To increase further the efficiency of transduction, we have combined LipofectAMINE and rAd-MFG-AP with the aim to enhance the uptake of viral particles into the target cells. The percentage of CD3+ cells transduced by rAd-MFG-AP-LipofectAMINE complex was 24+/-4% (range, 20-35%) after incubation with IL-2 plus IL-7 and 22+/-4% (range, 18-32%) after incubation with II-2 plus IL-12. Forty-eight hours after the incubation with rAd-MFG-AP, the transduced T lymphocytes were subjected to fluorescence-activated cell sorting and fractionated into AP+ and AP- cell subpopulations. The AP+ cell fraction, comprising 96.8% of AP+ cells, was evaluated by FACScan analysis for T lymphocyte surface antigens. The immunophenotyping of the transduced T lymphocytes has shown that there was not a particular subtype of T lymphocytes more susceptible to rAd-MFG-AP transduction. In addition, the transgene expression did not modify T lymphocyte functions, as demonstrated by results obtained by cytotoxicity assay before and after rAd-MFG-AP-LipofectAMINE complex transduction. In conclusion, human T lymphocytes can be efficiently transduced, under clinically applicable conditions, by adenovirus-LipofectAMINE complex after 7 days of culture with IL-2 and IL-12 or IL-7.

In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors. The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene. DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim. The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells. The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ. In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs.

Using 98 plasma samples from cancer patients undergoing antineoplastic chemotherapy, we compared the activities of aspartate aminotransferase and alanine aminotransferase measured by two different methods, with and without the addition of pyridoxal-5'-phosphate to the assay medium. Pyridoxal-5'-phosphate caused an increase of 1 to 20 U/l in aspartate aminotransferase and alanine aminotransferase activity in 90 and 78 patient plasma samples, respectively. Increases of aspartate aminotransferase and alanine aminotransferase activity of more than 20 U/l were observed in 8 and 20 samples, respectively. In 8 cases, the increase in alanine aminotransferase activity was greater than 50 U/l, whereas a similar increase in aspartate aminotransferase activity was decreased in only 2 cases. The considerable pyridoxal-5'-phosphate activation in aminotransferase activity observed in the plasma of a significant number of patients suggests that the use of the method with pyridoxal-5'-phosphate is advisable for a correct measurement of the catalytic concentration of aminotransferases in the plasma of patients undergoing chemotherapy.

The effects of dimethylformamide (DMF) on hemostatic functions, especially on platelet activity, were examined both in vitro and in vivo in 15 workers exposed to DMF (27 mg/m3, median value). Twenty-eight control subjects who were not exposed to DMF, but comparable for age, anthropometric data, and smoking habits, were also studied. Workers exposed to DMF showed a decrease in the number of platelets and had longer coagulation times, probably due to a change caused by DMF on the membrane receptor of platelets and on the phospholipid components of the clotting system.

We studied the ultrastructural morphology and function of platelets collected by apheresis procedure with discontinuous flow using the Surge Pump Technique. The platelet concentrates obtained by this technique are free of erythrocyte and lymphocyte contamination. The platelet morphology as well as the platelet aggregation induced by ADP, adrenaline and collagen of platelet concentrates were similar to those observed in PRP before the apheresis. The response to the hypotonic shock of platelet concentrates was also normal, indicating membrane integrity and good platelet metabolism. These results show that platelet concentrates obtained by the Surge Pump Technique maintain their haemostatic effects and may be infused in thrombocitopenic patients, reducing the risk of alloimmunization related to the presence of erythrocytes and lymphocytes.

The effect of Dimethylformamide on platelet function and on clotting system has been studied on eight workers exposed to the product, widely used in industry as a solvent of acrylic resins. A significant reduction of the number of the platelets and a drawing out of PTT and PT has been noticed. Such informations has been explained as chronical peripheral use or as modification of phospholipid components induced by DMF instead of synthesis defect, as no significant variation of the fibrinogen and of AT III values, proteins synthetized by liver, has been observed. The platelet aggregation has pointed out, in vivo, a reduction of ADP aggregation in the exposed subjects; in vitro, increasing quantities have determined an increasing reduction of the primary ADP and adrenaline aggregation.