T regulatory cells mediate immunosuppresion by adenosine in peripheral blood, sentinel lymph node and TILs from melanoma patients.

T regulatory cells (Tregs), involved in tumour tolerance, can generate Adenosine by CD39/CD73 surface enzymes, which identify four Tregs subsets: CD39+CD73- nTregs, CD39+CD73+ iTregs, CD39-CD73+ oTregs and CD39-CD73- xTregs. In melanoma patients, increased Tregs levels are detected in peripheral blood (PB), sentinel lymph node (SLN) and tumour infiltrating lymphocytes (TILs), but Adenosine role was not investigated yet. We examined total Tregs and Adenosine subsets in PB, SLN and TILs from melanoma patients (n = 32) and PB from healthy donors (HD; n = 10) by flow cytometry. Total Tregs significantly increased in stage III-IV patients PB, in SLN and TILs, as compared to HD/stage I-II patients. Tregs subsets analyses showed that: 1) PB nTregs significantly increased in SLN and decreased in TILs; 2) iTregs significantly increased in stage III-IV patients PB and further significantly increased in SLN and TILs; 3) PB oTregs and xTregs significantly decreased in SLN and TILs. Patients clinical features did not significantly influence total Tregs, except SLN excision order. Results confirmed Tregs role in melanoma progression and indicate Adenosine generation as a novel escape mechanism, being nTregs and iTregs increased in PB/SLN/TILs.

Long-term suppression of EAE relapses by pharmacological impairment of epitope spreading.

BACKGROUND AND PURPOSE: Immune events sustaining dendritic cell (DC)-dependent epitope spreading (ES) are of key relevance to the development of relapses during multiple sclerosis (MS). Although no drugs are currently available to target ES, its inhibition would represent a major advancement in MS therapy. Inhibitors of the enzyme PARP-1 afford protection in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). These drugs epigenetically impair antigen presentation by DCs, but whether these drugs affect ES is unknown. Here, we investigated whether short-term treatments with these compounds would impair ES, thereby preventing EAE relapses. EXPERIMENTAL APPROACH: We used a model of relapsing EAE in SJL mice and also adopted in vivo and ex vivo models of DC-dependent T-cell polarization. The effect of PARP-1 inhibitors on ES was evaluated at the humoral and cellular level. KEY RESULTS: Short-term treatments with PARP-1 inhibitors during the acute phase of relapsing EAE of mice induced, at later times, more tolerogenic DCs, increased numbers of Treg cells and impairment of ES at the humoral and cellular level. These effects are followed by long-lasting reduction of relapse severity and incidence, although drug treatment had been discontinued for several weeks. PARP-1 inhibitors also induced tolerogenic DCs and increased Treg cells number and function in a model of ovalbumin immunization. CONCLUSIONS AND IMPLICATIONS: Our data emphasize the therapeutic potential of PARP-1 inhibitors in the treatment of relapsing-remitting MS and additional ES-driven autoimmune disorders.

Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Insights from the AMI-Florence 2 Study.

Reticulated platelets (RP) are newly-formed platelets with a greater mass, a residual amount of RNA and an increased prothrombotic potential. No studies investigating the association between RP and the risk of cardiovascular death in acute coronary syndrome (ACS) patients are available. In the frame of the AMI-Florence 2 study, we investigated RP in 229 (154 M/ 75 F) ACS patients (125 ST-elevation myocardial infarction [STEMI]; 104 Non-STEMI/Unstable Angina). RP were measured by using the Sysmex XE-2100 haematology analyzer and were expressed as the percentage of RP out of the total optical platelet count (immature platelet fraction; IPF) and as the percentage of RP highly fluorescent (H-IPF). At one-year follow-up, 22 out of 229 patients (9.6%) died from cardiovascular causes. Higher values of IPF (p=0.05) and H-IPF (p=0.006) were detected in dead compared to alive patients. A receiver operating characteristics curve analysis identified IPF ≥3.3% and H-IPF ≥0.9% as optimal cut-off values to predict cardiovascular death. At the multivariate model adjusted for the Global Registry of Acute Coronary Events (GRACE) risk score, the association between RP and cardiovascular death remained significant for both IPF [OR (95%CI) : 4.15 (1.24-13.91) p=0.02] and H-IPF [OR (95%CI): H-IPF 5.03 (1.38-18.38) p=0.01]. In conclusion, RP are independent predictors of cardiovascular death and may be useful in improving risk stratification for ACS patients. Future prospective studies to evaluate the role of RP in determining cardiovascular events are warranted.

Protective effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts.

To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD(+) and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD(+) and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion.

Use of CFDA-SE for evaluating the in vitro proliferation pattern of human mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitors retaining the capability to undergo multilineage differentiation, mostly towards all the mesodermal cellular lineages. MSC growing under standard conditions are composed of two main subpopulations with a characteristic distribution in the morphologic flow cytometric scatter: RS (recycling stem) cells (small, agranular) and m (mature) MSC (large, moderately granular cells). METHODS: MSC obtained from BM of healthy donors and expanded in culture were characterized by evaluating both the expression of conventional markers and differentiation potential. We used CFSE, a lipophilic dye that is taken up by cell membranes, to investigate separately the proliferative activity of RS cells and mMSC subsets. RESULTS: With flow cytometric analysis, RS cells and mMSC showed nearly the same immunophenotypic pattern, even if a significantly smaller percentage of RS cells expressed some of the classic mesenchymal Ag. The RS cell fraction was confirmed to have a higher proliferative potential and such a feature was particularly evident under certain culture conditions. DISCUSSION: CFSE has been shown as a reliable method for studying the proliferative activity of MSC subpopulations identified by flow cytometric analysis. The acquisition parameter strategy is crucial for the accuracy of the analysis.

Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086.

WEB-2086 -- an antagonist of platelet-activating factor receptor (PAFR) with known anti-inflammatory, antiangiogenic and antileukaemic properties -- also proved to inhibit the proliferation in human solid tumour cell lines of different histology, and with much higher efficacy than in normal fibroblasts. A detailed analysis of WEB-2086 anticancer activity was then performed focusing on breast adenocarcinoma MCF-7 and MDA-MB-231 cells. WEB-2086-treated cells, either expressing (MCF-7) or unexpressing (MDA-MB-231) the oestrogen receptor (ER)alpha, underwent a dose-dependent growth arrest (IC(50)=0.65+/-0.09 and 0.41+/-0.07 mM, respectively) and accumulation in G(0)-G(1) phase. WEB-2086 also induced morphological and functional changes typical of mature mammary phenotype including (i) cell enlargement and massive neutral lipid deposition (best accomplished in MCF-7 cells); (ii) decrease in motility and active cathepsin D levels (mainly observed in highly invasive MDA-MB-231 cells). The expression of ERalpha was neither increased nor reactivated in treated MCF-7 or MDA-MB-231 cells, respectively. WEB-2086-induced differentiation in breast cancer cells involved the upregulation of PTEN, a key tumour suppressor protein opposing tumorigenesis, and was apparently independent of p53, PAFR, peripheral benzodiazepine receptor and ERalpha status. Overall, WEB-2086 can be proposed as an effective antiproliferative and differentiative agent with interesting translational opportunities to treat breast cancers in support to conventional chemotherapy.

VEGF inhibition and cytotoxic effect of aplidin in leukemia cell lines and cells from acute myeloid leukemia.

BACKGROUND: Aplidine (APL) is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that is under clinical phase II development. In contrast to the lack of bone marrow toxicity reported in phase I/II studies, it has been shown to induce cytotoxicity at very low concentration against lymphoblastic leukemia blast, as well as having an impact in the vascular endothelial growth factor (VEGF)/VEGF receptor 1 loop. PATIENTS AND METHODS: To confirm these findings we investigated APL-related VEGF inhibition and its cytotoxic effect on myeloid leukemic cells lines (K-562, HEL and HL60) and fresh leukemia blasts derived from 30 patients with acute myeloid leukemia (AML). The conventional active 4-demetoxi-daunorubicin (idarubicin; IDA) was included as a positive control. RESULTS: APL was found to be significantly (P<0.001) more active than IDA in obtaining 50% growth-inhibition in K-562, HEL and HL60 cell lines. Results obtained with AML blast cells were super imposible. ID(50) ranged from 0.024 to 0.610 microM for IDA (0.200+/-0.176) and from 0.001 to 0.108 microM for APL (0.020+/-0.031). Annexin V tests and cell cycle analysis performed on cell lines confirmed the stronger citotoxic capability of APL as apoptotic inducer and as a G(1) blocker. The inhibitory effects of APL on VEGF release and secretion have been confirmed by ELISA tests performed on HEL: the VEGF concentration in cell surnatant was reduced from 169 to 36 pg/ml after 24 h of exposure to a pharmacological concentration of APL. CONCLUSIONS: APL harbors a strong in vitro antileukemic activity at a concentration achievable in patients at non-myelotoxic doses. Our data also support the notion of an impact on VEGF secretion. Clinical studies with this new marine-derived compound in relapsed/resistant leukemia are underway.

Selective ex vivo expansion of cytomegalovirus-specific CD4+ and CD8+ T lymphocytes using dendritic cells pulsed with a human leucocyte antigen A*0201-restricted peptide.

Adoptive transfer of ex vivo-generated cytomegalovirus (CMV)-specific T lymphocytes may be effective in preventing CMV disease in allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We developed a procedure for expansion of CMV-specific T lymphocytes based on the antigen-presenting function of donor dendritic cells (DCs), pulsed with a human leucocyte antigen A*0201-restricted pp65 nonamer peptide. CMV-specific T lymphocytes were identified following induction of interferon gamma (IFN-gamma) secretion prompted by peptide exposure. Both CD8+ and CD4+ CMV-specific T lymphocytes were selectively produced in these cultures and showed CMV-restricted cytotoxicity. The simultaneous and selective expansion of CD4+ and CD8+ CMV-specific lymphocytes might be instrumental for more efficient in vivo function of infused CMV-specific lymphocytes.

Ligands of peroxisome proliferator-activated receptor gamma modulate profibrogenic and proinflammatory actions in hepatic stellate cells.

BACKGROUND & AIMS: Proliferation and migration of hepatic stellate cells (HSCs) and expression of chemokines are involved in the pathogenesis of liver inflammation and fibrogenesis. Peroxisome proliferator-activated receptor (PPAR)-gamma is a receptor transcription factor that controls growth and differentiation in different tissues. We explored the effects of PPAR-gamma agonists on the biological actions of cultured human HSCs. METHODS: HSCs were isolated from normal human liver tissue and used in their myofibroblast-like phenotype or immediately after isolation. Activation of PPAR-gamma was induced with 15-deoxy-Delta(12, 14)-prostaglandin J(2) or with troglitazone. RESULTS: PPAR-gamma agonists dose-dependently inhibited HSC proliferation and chemotaxis induced by platelet-derived growth factor. This effect was independent of changes in postreceptor signaling or expression of c-fos and c-myc and was associated with inhibition of cell cycle progression beyond the G(1) phase. Activation of PPAR-gamma also resulted in a complete inhibition of the expression of monocyte chemotactic protein 1 at the gene and protein levels. Comparison of quiescent and culture-activated HSCs revealed a marked decrease in PPAR-gamma expression in activated cells. CONCLUSIONS: Activation of PPAR-gamma modulates profibrogenic and proinflammatory actions in HSCs. Reduced PPAR-gamma expression may contribute to confer an activated phenotype to HSCs.

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.

We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568)

Induction of apoptosis by monosaccharide butyrate stable derivatives in chronic lymphocytic leukemia cells.

BACKGROUND AND OBJECTIVE: Different therapeutic approaches are needed to restore apoptotic mechanisms in CLL cells, as present ones are not successful. We assessed the apoptotic effects of stable butyrate derivatives on CLL lymphocytes: in these molecules a mannose molecule is bound as ester to one-five butyrate moieties, conferring pharmacological stability to the pro-drugs which are able to induce apoptosis in primary AML blasts. DESIGN AND METHODS: Peripheral blood samples obtained from 17 patients with typical B-CLL were cultured in the presence of 0.5-1mM D1 (O-n-butanoyl-2, 3-O-isopropylidene-a-D-mannofuranoside), F1 (1-O-n-butanoyl-2, 3-O-isopropylidene-D,L-xylitol) and G1 (1-O-n-butanoyl-D,L-xylitol) derivatives for 4 days and equimolar sodium butyrate as comparison. After culture, apoptosis was evaluated by cell morphology, cellular DNA content, pattern of DNA fragmentation, annexin V exposure on cell membrane, and cell cycle parameters. Bcl2, bax, and fas oncogene expression were also evaluated by the APAAP method. RESULTS: The addition to cell cultures of D1 or F1 or G1 butyrate monosaccharides as well as sodium butyrate 0.5 and 1 mM determined to different extents an increase in the percentage of apoptotic cells in all CLL samples, relatively to the method and butyrate molecule added in culture. Heterogeneity in CLL cell sensitivity to the three butyrates was observed. Up to 60-68% apoptotic bodies were present in treated cultures after exposure to D1 0.5-1 mM, 60-72% after F1 0.5-1 mM and 48-60% after G1 0.5-1 mM. Comparison of untreated versus treated cultures yielded important significance (p< 0.001). At DNA content analysis, analyzed by flow cytometry, apoptotic events were accounting for up to 70-77% of D1-treated and 68-74% of F1-treated CLL cells at 0.5 and 1 mM concentrations (p= 0. 0001, vs controls 0-39%), and for 72-81% of G1 (0.5-1 mM) treated cells (overall, p=0.005). Cell cycle parameters were not altered by addition of butyrates, but expression of Annexin V was greatly enhanced. In a limited number of CLL cases fas, bcl2/bax ratio was analyzed and found unmodified. INTERPRETATION AND CONCLUSIONS: Monosaccharide butyrate stable derivatives are potent inducers of primary CLL cell apoptosis, both in untreated and alkylating agent pre-treated cases. Our results suggest that the apoptotic pathways elicited by butyrate in CLL lymphocytes are direct, specific and most probably do not involve bcl2/bax. Pro-apoptotic agents like the stable monosaccharide butyrate derivatives here studied could bring more insights into CLL biology and resistance to apoptosis, and possibly originate alternative treatments for CLL.

Coexpression of erythroid and megakaryocytic genes in acute erythroblastic (FAB M6) and megakaryoblastic (FAB M7) leukaemias.

There is evidence to suggest a close relationship between the erythroid and megakaryocytic lineages. Using RT-PCR, we evaluated the coexpression of erythroid and megakaryocytic genes in blasts from 25 acute myeloid leukaemia (AML) cases (FAB M1-M7) and three unclassifiable leukaemias with trilineage dysplasia (trilineal AML). All FAB M6 and M7 and trilineal leukaemias expressed mRNAs for alpha-globin, glycoprotein IIb (GpIIb), erythropoietin receptor (Epo-R) and thrombopoietin receptor (c-mpl), but not for myeloperoxidase (MPO) which in contrast was expressed in the other FAB-subtype leukaemias. These data support the hypothesis that blasts from M7 and M6 leukaemias may derive from (or represent) a common progenitor cell with resident bipotentiality towards the megakaryocytic and erythrocytic lineages.

Erythropoietin upregulates the expression of its own receptor in TF-1 cell line.

In the erythroleukemia cell line TF-1, recombinant human erythropoietin (rHEpo), but not c-kit ligand, enhanced the number of cells expressing the erythropoietin receptor (EpoR), as measured by flow-cytometric analysis of binding of the biotin-labeled Epo. Moreover, 125I-Epo binding and Scatchard analyses, indicated that TF-1 cells, maintained in standard conditions with IL-3, and those stimulated with c-kit ligand, bear a single class of EpoR. On the other hand, cells cultured in the presence of rHEpo had a higher number of receptors than IL-3 or c-kit ligand-stimulated cells, and had two binding sites with different affinities for the ligand. EpoR mRNA expression was higher in cells exposed to rHEpo than in IL-3 or c-kit-stimulated cells. This difference may have been dependent on either a higher level of transcription or an increased stability of mRNA. The observed changes of EpoR in rHEpo-stimulated TF-1 cell line could cooperate, together with the alteration of the gene (3' end deletion), in the occurrence of the erythroleukemic process. Changes induced in EpoR by rHEpo were not accompanied by an increase in the expression of glycophorin A or globin chain mRNAs. This may suggest that rHEpo is unable to induce erythroid differentiation in TF-1 cells. The results also indicate that this cell line could be a model for the investigation of the role of transcription factor(s) in the expression of EpoR, and for the study of the mechanism(s) underlying the changes in the number and affinity of the cell receptors.

[The prevalence and clinical characteristics of heart failure in a population sample of Calabria]. / Prevalenza e caratteristiche cliniche dell'insufficienza cardiaca in un campione di popolazione della Calabria.

Two-hundred and fourteen patients with congestive heart failure were identified over a six-month period in the general practice of 29 GPs covering an adult population of 29,959 subjects residing in the region of Calabria, in southern Italy, with an overall prevalence of 7 per 1000. Males represented 52% of the cases and females 48%, with a median age of 75 years. On average, the condition was first diagnosed 41 months before the present examination. Patients generally had a high body mass index (28 kg/m2). Patients were classified as follows in the NYHA classification: 9.4% in class I, 45.3% in class II, 39.2% in class III, 6.1% in class IV. Hypertension, either alone or associated with ischemic heart disease (totally about 75% of cases), was the most common etiology, while COPD was the most commonly associated chronic condition. Clinical symptoms and signs were used to classify patients in a simplified version of the Boston score which was reported in 48% of cases as definite, 12% as possible, 6% as improbable and 34% as absent. A specific treatment was already ongoing in 97% of patients. The most commonly administered drugs were diuretics (83%), ACE-inhibitors (77%), and digitalis (67%). This three-drug combination (alone or with other drugs) covered 46% of patients. A comparison of four predefined typologies of treatment against the Boston score suggested that at least part of the outcome in classifying patients using this procedure was due to pathomorphosis of the syndrome induced by early pharmacological treatment.

Idarubicin in induction treatment of acute myeloid leukemia in the elderly.

BACKGROUND AND OBJECTIVE: The best approach to treatment of acute myeloid leukemia (AML) in elderly patients remains controversial. Intensive chemotherapy is the treatment of choice in selected patients, but age related changes might affect the pharmacokinetics of antineoplastic agents resulting in enhanced toxicity. We report our experience in elderly patients treated with idarubicin at attenuated doses plus cytarabine and etoposide. METHODS: Sixty-six AML patients, median age 66, with progressive disease and high tumor burden received idarubicin 8 mg/sqm i.v. d 1,3,5; cytarabine 200 mg/sqm by continuous i.v. infusion d 1-7; etoposide 60 mg/sqm i.v. d 1-5. A second course with the same drugs was planned irrespective of complete remission (CR) achievement. No consolidation was given; 44% had a documented preexisting myelodysplasia, 45% had a documented preexisting myelodysplasia, 45% presented with fever. Promyelocytic leukemias were excluded. RESULTS: Thirty-five patients (53%) achieved CR and 9 PR for an overall response rate of 67%. Nine of them (13%) died early or during the aplastic phase. Preexisting myelodysplasia had no significant impact on CR achievement. Resistant disease was associated with CD7 phenotype and unfavorable karyotype. Overall survival and disease free survival were 14 and 13 months, respectively. The major toxicity consisted of infectious complications (WHO > 2 in 24% of patients). Six patients died for infection, 2 for heart failure, 1 for pulmonary embolism. INTERPRETATION AND CONCLUSIONS: This induction regimen with attenuated doses of idarubicin is feasible and effective, but long-term survival remains an unresolved problem. Alternative post remission approaches are advisable in the aim of improving the remission duration.

Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro.

Incubation in severe hypoxia (1% oxygen) increased the number of erythroid bursts generated from full-term CD34+, or premature mononucleated, human cord blood (CB) cells, in semisolid cultures containing stem cell factor (SCF), interleukin (IL)-3 and erythropoietin (EPO). Severe hypoxia also enhanced the maintenance of erythroid burst-forming units (BFU-E) in CB cell liquid cultures. These positive effects of hypoxia on the maintenance and cloning efficiency of BFU-E did not extend to the other progenitors assayed. Hypoxia, on the other hand, markedly reduced the size and level of hemoglobinization of bursts and, in liquid cultures, suppressed the growth factor-stimulated numerical increase in BFU-E and inhibited the expression of CD36, a marker of erythroid colony-forming units and maturing erythroid precursors. However, when transferred to clonal assays incubated in air, cells from liquid cultures incubated in hypoxia or in air generated fully expanded and hemoglobinized bursts, suggesting that in hypoxia the clonogenic potential of BFU-E was maintained and the development of erythroid clones reversibly inhibited. These results indicate that hypoxia inversely regulates two subsequent phases of erythropoiesis, i.e., it enhances the maintenance of BFU-E and the early development of erythroid clones but inhibits the terminal expansion and maturation of these clones. The cloning of CB cells selected for CD34 positivity, when compared with that of the total population of mononucleated CB cells, revealed that the early development of erythroid bursts was either hypoxia-enhanced or hypoxia-insensitive, reflecting the existence of two different types of BFU-E. Hypoxia-enhanced BFU-E are relatively immature, are maintained in hypoxia but not in air, and account for a large part of CD34+ BFU-E and for a high percentage of the BFU-E in premature CB. Hypoxia-insensitive BFU-E are mostly CD34- and are largely predominant in full-term CB, and most probably correspond to a more mature type of BFU-E.

[Long-term captopril therapy in asymptomatic patients with severe chronic aortic insufficiency]. / Terapia a lungo termine con captopril in pazienti asintomatici con insufficienza aortica cronica di grado severo.

BACKGROUND: Clinical history of patients with severe chronic aortic regurgitation is characterized by a long period without any symptom, although the left ventricle enlarges progressively. The administration of oral vasodilators could reduce ventricular enlargement or its progression, delaying the development of myocardial dysfunction and/or the need for valvular surgery. OBJECTIVES: To verify the efficacy of long-term captopril therapy to reduce left ventricular mass and dimensions in patients with severe chronic aortic regurgitation. METHODS: This is a prospective echocardiographic study in which each individual patient is considered his own control case. Eleven asymptomatic patients with severe chronic aortic regurgitation in sinus rhythm, who had an ejection fraction greater than 50% and were not taking cardiovascular drugs, were orally administered captopril at the maximum tolerated dosage (127 +/- 13 mg/day). Follow-up lasted for 24 +/- 3 months. RESULTS: Left ventricular telediastolic diameter decreased from 69 +/- 5 to 61 +/- 3 mm (p < 0.01), telesystolic diameter decreased from 48 +/- 5 to 41 +/- 4 mm (p < 0.01); ejection fraction increased from 56 +/- 4 to 61 +/- 3% (p < 0.001); myocardial mass decreased from 208 +/- 32 to 174 +/- 27 g/m2 (p < 0.01), and mean wall stress from 264 +/- 35 to 203 +/- 25 mmHg (p < 0.001). All variations were still significant at 6 months. CONCLUSIONS: These results suggest that captopril has a favourable effect on left ventricular mass, dimensions and load conditions, and could favourably influence the natural history of chronic aortic regurgitation. The efficacy of medical treatment can be verified through serial echocardiographic study.

Medical treatment of end-stage heart failure.

Congestive heart failure is a lethal condition that affects an increasing number of patients. In recent years a great amount of data have accumulated on the pathophysiology and medical and surgical therapy of this condition. In spite of the advances in its management and the great number of patients affected, common errors are still made by internists and cardiologists in the use of drugs and therapeutic strategies. Digitalis has only recently been shown to affect hemodynamics, exercise capacity, and clinical symptoms, but the effects on survival still have to be demonstrated. Loop diuretics, eventually combined with thiazides and antialdosterone drugs in patients with clinical signs and symptoms of fluid retention, are the mainstays of therapy of congestive heart failure. In order to make diuretic therapy efficacious, moderate salt and water intake restriction is mandatory. Angiotensin-converting enzyme (ACE) inhibitors are now considered unavoidable drugs in the management of heart failure, and an attempt to reach the doses that have been shown to be efficacious for survival in the large trials has to be made in every patient with this condition. Other vasodilators, such as hydralazine and nitrates, which show a less pronounced effect on survival but more effective hemodynamic actions than ACE inhibitors, may be used to control mitral insufficiency or to improve hemodynamics in very sick patients. Hemodynamic instability refractory to increasing doses of vasodilators and diuretics is a severe condition that requires hospital admission to administer drugs parenterally. These patients are usually treated with the combination of catecholamines and phosphodiesterase inhibitors associated with intravenous diuretics until clinical stability is again achieved and oral therapy is resumed and restructured. The use of aggressive pharmacological therapy and phosphodiesterase inhibitors has reduced the need for assisted circulatory support in these patients. Beta-blockers have shown promising results when administered to patients with heart failure, although a definitive demonstration of their effects on survival is still lacking. Other additional measures that need to be considered in patients with end-stage congestive heart failure are the use of antiarrhythmic drugs and anticoagulation.

Megakaryocyte-like increase in ploidy of Friend's erythroleukemia cells induced to endoreplication by colcemid.

Previous work from this laboratory has shown that Friend's murine erythroleukemia cells (MELCs) express some bio-chemical traits of the megakaryocytic lineage. The supposed mixed erythroid/megakaryocytic nature of these cells has been investigated further by challenging MELCs with the antimicrotubule agent colcemid. This compound, at the concentration of 40 nM, was found to induce a striking arrest of cell growth without significant effects on viability. At the same time, the bulk of treated MELCs underwent a large increase in size to contain, after 3 days, as much as 4 times more proteins and 5 times more DNA than controls. As shown by high rates of 3H-thymidine incorporation, increase in DNA content was the result of active synthesis without completion of intervening mitosis according to a process that closely resembled endoreplication. Eventually, colcemidinduced MELCs presented multilobed nuclei and were arranged into discrete ploidy groups containing up to 16 N levels of DNA. Moreover, upon colcemid addition, MELCs initiated a polyploid response that was shown to continue, even in the absence of the inducer, to yield cells that became strongly positive for acetylcholinesterase (AChE) in the late stages of culture. These effects were compatible with a colcemid-induced commitment of MELCs to megakaryocyte differentiation, for which these cells seemed to be definitely programmed. The expression of megakaryocyte features in MELCs provided further evidence for the bipotentiality (erythroid/megakaryocytic) of this model.

Defective aggregation in cirrhosis is independent of in vivo platelet activation.

BACKGROUND/AIMS: Platelet function abnormalities contribute to the hemostatic defect in patients with cirrhosis. In this study we evaluated the occurrence of in vivo platelet activation as a possible mechanism of defective platelet aggregation in patients with cirrhosis. METHODS: Nine patients with severe (Child B-C) cirrhosis and defective platelet aggregation were studied in comparison with age- and sex-matched healthy controls. The presence of activated platelets in the bloodstream was evaluated by fluorescence-activated flow cytometry using antibodies directed against activation-dependent platelet proteins and by measuring plasma levels of beta-thromboglobulin and platelet factor 4. Urinary levels of 11-dehydro-TXB2 and of 2,3-dinor-TXB2 were assayed by radioimmunoassay following chromatographic separation. RESULTS: In unstimulated platelets, the expression of both GMP 140 and GP 53 was not significantly different in patients with cirrhosis and in controls. After stimulation with ADP and epinephrine, expression of activation-dependent antigens was lower in platelets from patients (GMP 140: 0.64 +/- 0.09 vs 0.73 +/- 0.04, p = 0.02; GP 53: 0.41 +/- 0.13 vs 0.54 +/- 0.14). Plasma levels of beta-thromboglobulin and platelet factor 4, as indexes of in vivo platelet activation, were also comparable in the two groups of subjects. Urinary levels of 11-dehydro-TXB2 and of 2,3-dinor-TXB2, the major systemic metabolites of TXA2, were significantly higher in patients with cirrhosis (1807 +/- 518 vs 341 +/- 121 ng/pg creatinine and 693 +/- 512 vs 205 (93 ng/pg creatinine, respectively, p < 0.001). However, increased excretion of TXB2 metabolites was also observed in three patients with chronic autoimmune thrombocytopenia. CONCLUSIONS: These data indicate that circulating platelets are not activated in cirrhosis, and that defective aggregation is most likely dependent on the alteration of the transmembrane signaling pathways. The increased urinary excretion of systemic TXA2 metabolites may be related to increased intrasplenic platelet destruction.