Temporal Dynamics of 'Ca. Phytoplasma mali' Load in the Insect Vector Cacopsylla melanoneura.

The transmission of phytoplasmas is the result of an intricate interplay involving pathogens, insect vectors and host plants. Knowledge of the vector's competence during its lifespan allows us to define more sustainable well-timed control strategies targeted towards the most worrisome life stages. We investigated the temporal dynamics of 'Candidatus Phytoplasma mali' load in Cacopsylla melanoneura in the different developmental stages in Northwest Italy. The phytoplasma load in the vector was evaluated in overwintering adults, nymphs and newly emerged adults after different acquisition access periods. Moreover, we followed the multiplication of the phytoplasma during the aestivation and the overwintering period on conifers. Our results confirmed the ability of remigrants to retain the phytoplasma until the end of winter. We also highlighted the high acquisition efficiency and vector competence, based on phytoplasma load, of nymphs and newly emerged adults. Therefore, particular attention should be paid to the management of overwintered C. melanoneura as soon as they return to the orchards, but also to newly emerged adults, particularly in orchards with a high infection rate and when the migration to conifers is delayed.

EvaGreen real-time PCR protocol for specific 'Candidatus Phytoplasma mali' detection and quantification in insects.

In this paper the validation and implementation of a Real-time PCR protocol based on ribosomal protein genes has been carried out for sensitive and specific quantification of 'Candidatus (Ca.) Phytoplasma mali' (apple proliferation phytoplasma, APP) in insects. The method combines the use of EvaGreen(®) dye as chemistry detection system and the specific primer pair rpAP15f-mod/rpAP15r3, which amplifies a fragment of 238 bp of the ribosomal protein rplV (rpl22) gene of APP. Primers specificity was demonstrated by running in the same Real-time PCR 'Ca. Phytoplasma mali' samples with phytoplasmas belonging to the same group (16SrX) as 'Ca. Phytoplasma pyri' and 'Ca. Phytoplasma prunorum', and also phytoplasmas from different groups, as 'Ca. Phytoplasma phoenicium' (16SrIX) and Flavescence dorée phytoplasma (16SrV). 'Ca. Phytoplasma mali' titre in insects was quantified using a specific approach, which relates the concentration of the phytoplasma to insect 18S rDNA. Absolute quantification of APP and insect 18S rDNA were calculated using standard curves prepared from serial dilutions of plasmids containing rplV-rpsC and a portion of 18S rDNA genes, respectively. APP titre in insects was expressed as genome units (GU) of phytoplasma per picogram (pg) of individual insect 18S rDNA. 'Ca. Phytoplasma mali' concentration in examined samples (Cacopsylla melanoneura overwintered adults) ranged from 5.94 × 10(2) to 2.51 × 10(4) GU/pg of insect 18S rDNA. Repeatability and reproducibility of the method were also evaluated by calculation of the coefficient of variation (CV%) of GU of phytoplasma and pg of 18S rDNA fragment for both assays. CV less than 14% and 9% (for reproducibility test) and less than 10 and 11% (for repeatability test) were obtained for phytoplasma and insect qPCR assays, respectively. Sensitivity of the method was also evaluated, in comparison with conventional 16S rDNA-based nested-PCR procedure. The method described has been demonstrated reliable, sensitive and specific for the quantification of 'Ca. Phytoplasma mali' in insects. The possibility to study the trend of phytoplasma titre in the vectors will allow a deepen investigation on the epidemiology of the disease.

The in vitro effects of verbascoside on human platelet aggregation.

Preliminary in vitro and animal studies have shown that verbascoside, a phenolic compound, may have several favourable biological activities, including an influence on endothelial function and on platelet aggregation. We sought to evaluate the effects of verbascoside, biotechnologically produced from plant cell cultures, on human platelet aggregation (PA). The blood from 40 aspirin-naïve volunteers with at least one cardiovascular risk factor was preincubated in vitro with verbascoside (1 and 2 mg/dL) and aspirin (100 µM). The blood from 20 patients with a prior diagnosis of coronary heart disease who were chronically assuming aspirin was preincubated in vitro with verbascoside (1 and 2 mg/dL). PA is measured with a light transmission aggregometry and multiplate analyzer. As compared to reference, preincubation with verbascoside resulted in a significant inhibition of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced PA (p < 0.01 for both). Verbascoside 2 mg/dL did not show a stronger effect as compared to verbascoside 1 mg/dL (p = 0.4). As expected, the in vitro addition of aspirin reduced AA induced PA (p < 0.01), but not that induced by ADP (p = 0.5). The addition of verbascoside to the blood of aspirin-treated patients did not improve the values of PA after AA stimulus (p = 0.8), whereas it ensured a stronger inhibition after ADP stimulus (p < 0.01). Verbascoside in vitro affects PA by mildly inhibiting aggregation, triggered both by ADP and AA. These preliminary data, while intriguing, require confirmation in subjects receiving verbascoside orally in order to determine whether these findings are clinically relevant.

Prasugrel versus tirofiban bolus with or without short post-bolus infusion with or without concomitant prasugrel administration in patients with myocardial infarction undergoing coronary stenting: the FABOLUS PRO (Facilitation through Aggrastat By drOpping or shortening Infusion Line in patients with ST-segment elevation myocardial infarction compared to or on top of PRasugrel given at loading dOse) trial.

OBJECTIVES: The authors sought to compare the effect on inhibition of platelet aggregation (IPA) of prasugrel therapy versus tirofiban bolus with or without a post-bolus short drug infusion in ST-segment elevation myocardial infarction (STEMI) patients. BACKGROUND: The degree and rapidity of IPA after prasugrel alone with or without concomitant glycoprotein IIb/IIIa inhibition in STEMI patients is unknown. METHODS: A total of 100 STEMI patients randomly received prasugrel 60 mg versus 25 µg/kg tirofiban bolus with or without post-bolus 2-h infusion of tirofiban, with or without concomitant prasugrel. IPA at light transmission aggregometry was performed throughout 24 h. The primary endpoint was IPA stimulated with 20 µmol/l adenosine diphosphate (ADP) at 30 min. RESULTS: At 30 min, patients in the prasugrel group showed a significantly lower IPA to 20 µmol/l ADP stimulation as compared with tirofiban-treated patients (36 ± 35 vs. 87 ± 31, p < 0.0001). Similarly, patients taking prasugrel showed a suboptimal degree of platelet inhibition for at least 2 h compared with tirofiban patients. Post-bolus tirofiban infusion was necessary to maintain a high level of IPA beyond 1 h after bolus administration if concomitant clopidogrel was given, whereas the bolus-only tirofiban and concomitant prasugrel led to the higher and more consistent IPA levels after both ADP and thrombin receptor-activating peptide stimuli than either therapy alone. CONCLUSIONS: Our study shows that prasugrel administration leads to a suboptimal IPA for at least 2 h in STEMI patients. Yet, prasugrel, given in association with a bolus only of glycoprotein IIb/IIIa inhibitor, obviates the need of post-bolus infusion and almost completely abolishes residual variability of IPA after treatment. (Facilitation through Aggrastat By drOpping or shortening Infusion Line in patients with ST-segment elevation myocardial infarction compared to or on top of PRasugrel given at loading dOse [The FABOLUS PRO trial]; NCT01336348).

Short- versus long-term duration of dual-antiplatelet therapy after coronary stenting: a randomized multicenter trial.

BACKGROUND: The optimal duration of dual-antiplatelet therapy and the risk-benefit ratio for long-term dual-antiplatelet therapy after coronary stenting remain poorly defined. We evaluated the impact of up to 6 versus 24 months of dual-antiplatelet therapy in a broad all-comers patient population receiving a balanced proportion of Food and Drug Administration-approved drug-eluting or bare-metal stents. METHODS AND RESULTS: We randomly assigned 2013 patients to receive bare-metal, zotarolimus-eluting, paclitaxel-eluting, or everolimus-eluting stent implantation. At 30 days, patients in each stent group were randomly allocated to receive up to 6 or 24 months of clopidogrel therapy in addition to aspirin. The primary end point was a composite of death of any cause, myocardial infarction, or cerebrovascular accident. The cumulative risk of the primary outcome at 2 years was 10.1% with 24-month dual-antiplatelet therapy compared with 10.0% with 6-month dual-antiplatelet therapy (hazard ratio, 0.98; 95% confidence interval, 0.74-1.29; P=0.91). The individual risks of death, myocardial infarction, cerebrovascular accident, or stent thrombosis did not differ between the study groups; however, there was a consistently greater risk of hemorrhage in the 24-month clopidogrel group according to all prespecified bleeding definitions, including the recently proposed Bleeding Academic Research Consortium classification. CONCLUSIONS: A regimen of 24 months of clopidogrel therapy in patients who had received a balanced mixture of drug-eluting or bare-metal stents was not significantly more effective than a 6-month clopidogrel regimen in reducing the composite of death due to any cause, myocardial infarction, or cerebrovascular accident. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00611286.

The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride.

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecific mycelial combat. The metabolite profiles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium influx in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant γ-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium influx in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxification of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments.

Genetic determinants of on-clopidogrel high platelet reactivity.

Clopidogrel has been used (alone or in association with aspirin) to prevent vascular complications in atherothrombotic patients, to prevent stent thrombosis (ST) in patients undergoing percutaneous coronary intervention (PCI) and as a long-term prevention of cardiovascular and cerebrovascular events. Unfortunately, it is important to note that there are a number of patients who, during clopidogrel therapy, show and maintain a high platelet reactivity (PR), similar to that observed before the start of antiplatelet therapy. Clopidogrel pro-drug is absorbed in the intestine and this process is influenced by P-glycoprotein-1 (P-GP). Its conversion into 2-oxo clopidogrel is regulated by cytochromes (CYP) called CYP2C19, CYP2B6 and CYP1A2. Whereas, the final transformation into the active metabolite is regulated by CYP called CYP2C19, CYP2C9, CYP2B6, CYP3A4, CYP3A5 and, as recently emerged, by the glycoprotein paraoxonase-1 (PON1). The genes encoding these enzymes are characterized by several polymorphisms. Some of these are able to modify the activity of proteins, reducing the concentration of active metabolite and the values of on-clopidogrel PR. Only one gene polymorphism (CYP2C19*17) increases the clopidogrel metabolization and so the clopidogrel-induced platelet inhibition. Several studies have clearly associated these gene polymorphisms to both ischemic and bleeding complications in patients receiving dual antiplatelet therapy. The aim of this review is to describe the principal gene polymorphisms influencing on-clopidogrel PR and their relationship with long-term clinical outcome.

Prediction of 1-year clinical outcomes using the SYNTAX score in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention: a substudy of the STRATEGY (Single High-Dose Bolus Tirofiban and Sirolimus-Eluting Stent Versus Abciximab and Bare-Metal Stent in Acute Myocardial Infarction) and MULTISTRATEGY (Multicenter Evaluation of Single High-Dose Bolus Tirofiban Versus Abciximab With Sirolimus-Eluting Stent or Bare-Metal Stent in Acute Myocardial Infarction Study) trials.

OBJECTIVES: This study sought to evaluate the impact of SYNTAX score (SXscore), and compare its performance in isolation and combination with the PAMI (The Primary Angioplasty in Myocardial Infarction Study) score, for the prediction of 1-year clinical outcomes in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention. BACKGROUND: Patients with STEMI were excluded from the original SYNTAX score (SXscore) algorithm. Therefore, the utility of using the SXscore in this patient group remains undefined. METHODS: SXscore was calculated retrospectively in 807 patients with STEMI enrolled in the randomized STRATEGY (Single High-Dose Bolus Tirofiban and Sirolimus-Eluting Stent Versus Abciximab and Bare-Metal Stent in Acute Myocardial Infarction) and MULTISTRATEGY (Multicenter Evaluation of Single High-Dose Bolus Tirofiban Versus Abciximab With Sirolimus-Eluting Stent or Bare-Metal Stent in Acute Myocardial Infarction Study) clinical trials. Clinical outcomes of all-cause death, reinfarction, and clinically driven target vessel revascularization were subsequently stratified according to SXscore tertiles: SX(LOW) ≤ 9 (n = 311), 9 < SX(MID) ≤ 16 (n = 234), SX(HIGH) >16 (n = 262). RESULTS: At 1-year follow-up, all clinical outcomes including mortality, mortality/reinfarction, major adverse cardiac events (MACE) (a composite of all-cause death, reinfarction and target vessel revascularization), and definite, definite/probable, and any stent thrombosis were all significantly higher in patients in the highest SXscore tertile. SXscore was identified as an independent predictor of mortality, MACE, and stent thrombosis out to 1-year follow-up. The combination SYNTAX-PAMI score led to a net reclassification improvement of 15.7% and 4.6% for mortality and MACE, respectively. The C-statistics for the SXscore, PAMI score, and the combined SYNTAX-PAMI score were 0.65, 0.81, and 0.73 for 1-year mortality, and 0.68, 0.64, and 0.69 for 1-year MACE, respectively. CONCLUSIONS: SXscore does have a role in the risk stratification of patients with STEMI having primary percutaneous coronary intervention; however, this ability can be improved through a combination with clinical variables. (Multicentre 2×2 Factorial Randomised Study Comparing Tirofiban Versus Abciximab and SES Versus BMS in AMI; NCT00229515).

Randomized comparison of 6- versus 24-month clopidogrel therapy after balancing anti-intimal hyperplasia stent potency in all-comer patients undergoing percutaneous coronary intervention Design and rationale for the PROlonging Dual-antiplatelet treatment after Grading stent-induced Intimal hyperplasia study (PRODIGY).

BACKGROUND: The optimal duration of clopidogrel therapy after coronary stenting is debated because of the scarcity of randomized controlled trials and inconsistencies arising from registry data. Although prolonged clopidogrel therapy after bare metal stenting is regarded as an effective secondary prevention measure, the safety profile of drug-eluting stents itself has been questioned in patients not receiving ≥ 12 months of dual-antiplatelet therapy. HYPOTHESIS: Twenty-four months of clopidogrel therapy after coronary stenting reduces the composite of death, myocardial infarction, or stroke compared with 6 months of treatment. STUDY DESIGN: PRODIGY is an unblinded, multicenter, 4-by-2 randomized trial. All-comer patients with indication to coronary stenting are randomly treated-balancing randomization-with bare metal stent (no active late loss inhibition), Endeavor Sprint zotarolimus-eluting stent (Medtronic, Santa Rosa, CA) (mild late loss inhibition), Taxus paclitaxel-eluting stent (Boston Scientific, Natick, MA) (moderate late loss inhibition), or Xience V everolimus-eluting stent (Abbott Vascular, Santa Clara, CA) (high late loss inhibition). At 30 days, patients in each stent group are randomly allocated to receive 24 or up to 6 months of clopidogrel therapy-primary end point randomization. With 1,700 individuals, this study will have >80% power to detect a 40% difference in the primary end point after sample size augmentation of 5% and a background event rate of 8%. SUMMARY: The PRODIGY trial aims to assess whether 24 months of clopidogrel therapy improves cardiovascular outcomes after coronary intervention in a broad all-comer patient population receiving a balanced mixture of stents with various anti-intimal hyperplasia potency.

Characterization of the intracellular signalling capacity of natural FXa mutants with reduced pro-coagulant activity.

INTRODUCTION: Factor X (FX) is a serine-protease playing a crucial role in the blood coagulation pathway and triggering intracellular signalling in a variety of cells via protease-activated receptors (PARs). By exploiting naturally occurring variants (V342A and G381D, catalytic domain; E19A, gamma-carboxyglutamic acid (GLA)-rich domain), we investigated the relationship between the pro-coagulant activity and the signal transduction capacity of FX. MATERIALS AND METHODS: Recombinant FX (rFX) variants were expressed in Human Embryonic Kidney cells and purified by immunoaffinity chromatography. Activated rFX (rFXa) variants were characterized for pro-coagulant, amidolytic and thrombin generation activity. rFXa signalling was assessed through evaluation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in C2C12 myoblasts. RESULTS AND CONCLUSIONS: rFX variants showed reduced (rFX-342A, 29%; rFX-19A, 12%) or not detectable (rFX-381D) amidolytic activity. Thrombin generation activity in a plasma system was also decreased either upon activation by Russell's viper venom (rFX-342A, 38%; rFX-19A, 7%; rFX-381D, not detectable) or by the extrinsic pathway (rFX-342A, 36%; rFX-19A, rFX-381D, not detectable). The rFXa-381D mutant displayed little or no enzymatic activity, and did not induce any appreciable signal transduction capacity. The rFXa-342A mutant induced a dose-dependent signalling with a 50% reduced signalling capacity. At the highest concentration (174 nM), signalling progressed with a time course similar to that of rFXa-wt. Zymogen rFX-19A showed defective and incomplete activation resulting in strongly reduced enzymatic activity and signalling. Taken together our data are consistent with a close correlation between pro-coagulant activity and intracellular signalling capacity.

Characterization of anti-coagulant properties of prenylated coumarin ferulenol.

We investigated the mechanisms underlying severe bleeding occurring upon consumption of Ferula communis. The prenylated coumarin ferulenol extracted from this plant did not directly affect blood coagulation but showed hepatocyte cytotoxicity and, at non-cytotoxic concentrations (<100 nM), impaired factor X biosynthesis (40% reduction). Studies with ferulenol derivatives indicated the prenyl residue as major determinant of ferulenol activity.

Molecular characterization of factor X deficiency associated with borderline plasma factor X level.

Borderline plasma factor X (FX) levels might complicate the diagnosis of FX deficiency. An asymptomatic individual with 73% FX activity was identified to be heterozygous for the Val342Ala mutation. Expression studies suggested that this substitution is responsible for a CRM+ FX variant with normal activation but modestly reduced catalytic function.