Rational design,preparation and characterization of recombinant Ag85B variants and their glycoconjugates with T-cell antigenic activity against Mycobacterium tuberculosis

Tuberculosis is the deadliest infectious disease in the world. The variable efficacy of the current treatments highlights the need for more effective agents against this disease. In the past few years, we focused on the investigation of antigenic glycoconjugates starting from recombinant Ag85B (rAg85B), a potent protein antigen from Mycobacterium tuberculosis. In this paper, structural modifications were rationally designed in order to obtain a rAg85B variant protein able to maintain its immunogenicity after glycosylation. Lysine residues involved in the main T-epitope sequences (namely, K30 and K282) have been substituted with arginine to prevent their glycosylation by a lysine-specific reactive linker. The effectiveness of the mutation strategy and the detailed structure of resulting neo-glycoconjugates have been studied by intact mass spectrometry, followed by peptide and glycopeptide mapping. The effect of K30R and K282R mutations on the T-cell activity of rAg85B has also been investigated with a preliminary immunological evaluation performed by enzyme-linked immunospotting on the different variant proteins and their glycosylation products. After glycosylation, the two variant proteins with an arginine in position 30 completely retain the original T-cell activity, thus representing adequate antigenic carriers for the development of efficient glycoconjugate vaccines against tuberculosis.

Recombinant Ag85B variants were designed and prepared to improve the immunogenicity of a potential glycoconjugate vaccine against tuberculosis.     

Comparison of antithrombotic strategies in patients with cryptogenic stroke and patent foramen ovale: an updated meta-analysis

Purpose

Patients with patent foramen ovale (PFO) and cryptogenic ischemic stroke (CS) are at risk for stroke recurrence. The optimal antithrombotic strategy in patients who undergo medical management is still debated.

Methods

We systematically searched the literature for studies that reported on cerebrovascular event recurrences and/or death in patients with PFO treated with oral anticoagulation (OAC) or antiplatelet therapy (APT) for secondary prevention of CS. The efficacy endpoints were stroke recurrence and the composite of stroke, transient ischemic attack or all-cause death. Major bleedings represented the safety endpoint.

Results

A total of 16 studies with 3953 patients (OAC?=?1527, APT?=?2426) were included. Weighted mean follow-up was 2.9 years. OAC was associated with a significant reduction in the risk of stroke compared with APT (RR 0.65; 95% CI 0.44–0.95; ARR 2%, NNT 49), while no difference was found regarding the composite outcome (RR 0.78; 95% CI 0.57–1.07) and the safety outcome (RR 1.57; 95% CI 0.85–2.90; p?=?0.15).

Conclusions

OAC was more effective than APT in reducing the risk of stroke recurrence in patients with PFO and CS, without a significant increase in the risk of major bleedings. Our findings support the need for further randomized data focused on the comparison of antithrombotic strategies in this setting.

     

Fluorescent cholangiography: An up-to-date overview twelve years after the first clinical application

Laparoscopic cholecystectomy (LC) is one of the most frequently performed gastrointestinal surgeries worldwide. Bile duct injury (BDI) represents the most serious complication of LC, with an incidence of 0.3%-0.7%, resulting in significant perioperative morbidity and mortality, impaired quality of life, and high rates of subsequent medico-legal litigation. In most cases, the primary cause of BDI is the misinterpretation of biliary anatomy, leading to unexpected biliary lesions. Near-infrared fluorescent cholangiography is widely spreading in clinical practice to delineate biliary anatomy during LC in elective and emergency settings. The primary aim of this article was to perform an up-to-date overview of the evolution of this method 12 years after the first clinical application in 2009 and to highlight all advantages and current limitations according to the available scientific evidence.     

Use of a monoclonal antibody against human heart ferritin for evaluating acidic ferritin concentration in human serum

Immunoassays for acidic ferritins rich in H subunits have shown that these isoferritins are predominant in some cells such as monocytes and red blood cells but have provided conflicting results about their presence in human serum. We have used an immunoradiometric assay based on a monoclonal antibody against human heart ferritin (monoclonal 2A4) for evaluating acidic ferritin concentration in human serum. This assay proved to be highly specific for acidic isoferritins having more than 60% H subunits. Heart-type ferritin was detected in only one fifth of normal sera and sera from patients with iron overload; values were very low compared with those for basic ferritin. Acidic ferritin was found in relatively high concentrations in most patients with iron deficiency anaemia. In other disease states characterized by increased serum concentrations of basic ferritin, acidic ferritin was always less than 21% of the total ferritin. Dialysis in low-ionic-strength buffer showed that both normal and pathological sera had binding factors for human heart ferritin. We conclude that: (i) human serum contains low concentrations of acidic isoferritins which, at variance with basic ferritin, do not appear to be directly related to the amount of storage iron; (ii) the findings of the present study reinforce the opinion that basic and acidic ferritins have different functional behaviours.     

Metabolic effects and disposition of sebacate, an alternate dicarboxylic fuel substrate.

Disodium sebacate is a 10-carbon-atom dicarboxylic acid, proposed as substrate for parenteral nutrition. We investigated its pharmacokinetic profile and thermogenic effect during a short-time infusion (5 h at 10 g/h) in 7 male volunteers. Sebacate in serum and urine was measured by high-performance liquid chromatography. A single-compartment model with two linear elimination routes was fitted. Metabolic measurements (VO2, VCO2, respiratory quotient, metabolic rate) were continuously performed for 8 h (5 h during and 3 h after the infusion) by a canopy indirect calorimeter. The apparent volume of distribution of sebacate was 8.39 +/- 0.69 liters, and the plasma fractional removal rate constant was 0.0086 +/- 0.00077 min-1. The average half-life and plasma clearance were 80.6 min and 72 ml/min, respectively. The increase in metabolic rate, the decrease in respiratory quotient and the changes in ketone body, glucagon and insulin levels during the infusion were not significant. 24-hour catecholamine excretion was within normal limits. Calories administered by sebacate seem to be available for utilization without relevant metabolic side effects.     

Favourable effects of heart rate reduction with intravenous administration of ivabradine in patients with advanced heart failure

BACKGROUND: Heart rate (HR) reduction may be useful in treatment of patients with heart failure (HF). There are no data on the haemodynamic effects of ivabradine (a selective I(f) current inhibitor) in advanced HF patients. AIMS: To assess the haemodynamic effects of ivabradine in patients with advanced HF and markedly depressed left ventricular (LV) function. METHODS AND RESULTS: Ten NYHA class III patients (50+/-12 years, LV ejection fraction 21+/-7%) underwent 24-h haemodynamic monitoring. Ivabradine 0.1 mg/kg was infused over 90', followed by 0.05-0.075 mg/kg in the subsequent 90'. Baseline HR was 93+/-8 bpm, cardiac index (CI) 2.2+/-0.6 l/min*m2; LV stroke volume 44+/-11 ml and systolic work 39+/-13 g. Ivabradine significantly reduced HR, by a maximum of 27% (to 68+/-9 bpm) at 4 h, without decreasing CI. Ivabradine increased stroke volume and LV systolic work by a maximum of 51% (to 66+/-17 ml) and 53% (to 58+/-20 g) at 4 h. No serious adverse events occurred. CONCLUSION: In patients with advanced HF and markedly depressed LV function, the acute administration of ivabradine is well tolerated, effectively reduces HR, markedly increases stroke volume and preserves cardiac output. Ivabradine appears a promising approach for the treatment of patients with moderate and advanced heart failure.