Francesco Cardona

Endotoxaemia modulates Toll-like receptors on leucocytes in humans

Summary. The modulation of Toll‐like receptors (TLR) 1, 2 and 4 was studied during experimental human endotoxaemia. Healthy volunteers received 2 ng/kg of lipopolysaccharide (LPS) endotoxin (n = 10). TLR1, 2 and 4 expression occurred on monocytes and neutrophils, with monocytes expressing higher baseline levels of TLR2. LPS infusion downmodulated TLR4 expression on neutrophils, with maximal downregulation occurring at 24 h (−62% from baseline; P < 0·03 versus baseline). Monocyte TLRs were upregulated in vivo (TLR1 and 2), and in vitro (TLR1, 2 and 4) 8 h after LPS bolus (P < 0·05 versus baseline). Therefore, neutrophils and monocytes differentially express surface TLRs, and endotoxaemia differentially regulates TLR expression.

A Randomized, Controlled Trial of the Effects of Adding Vitamin B12 and Folate to Erythropoietin for the Treatment of Anemia of Prematurity

BACKGROUND. Premature infants, especially those with birth weights of <1500 g, often suffer from anemia of prematurity and associated problems. Erythropoietin therapy is a safe effective way to prevent and to treat anemia of prematurity. We hypothesized that combined administration of vitamin B12 and folate with erythropoietin and iron would enhance erythropoietin-induced erythropoiesis. METHODS. In a randomized, controlled trial, 64 premature infants (birth weight: 801–1300 g) receiving erythropoietin and iron supplementation were assigned randomly to receive either vitamin B12 (3 μg/kg per day) and folate (100 μg/kg per day) (treatment group) or a lower dose of folate (60 μg/kg per day) (control group). RESULTS. During the 4-week observation period, vitamin B12 and folate enhanced erythropoietin-induced erythropoiesis significantly, as indicated by a 10% increase in red blood cell counts, compared with folate alone. Hemoglobin and hematocrit levels remained stable in the treatment group, whereas they decreased in the control group. Vitamin B12 levels in the treatment group increased over baseline and control values, whereas red blood cell folate levels were comparable between the groups. Subsequent analysis showed slight nonsignificant differences in baseline red blood cell count, hemoglobin level, hematocrit level, and mean corpuscular volume values, which must be addressed as a limitation. CONCLUSIONS. With the limitation of a slight imbalance in baseline data between the study groups, combined therapy with vitamin B12, folate, erythropoietin, and orally and intravenously administered iron seemed more effective in stimulating erythropoiesis among premature infants, compared with erythropoietin, iron, and low-dose folate alone. Additional trials are necessary to confirm these data.

Effects of a Combined Therapy of Erythropoietin, Iron, Folate, and Vitamin B12 on the Transfusion Requirements of Extremely Low Birth Weight Infants

OBJECTIVES. Erythropoietin is frequently administered to premature infants to stimulate erythropoiesis. The primary goal of erythropoietin therapy is to reduce transfusions, but the efficacy of erythropoietin has not been convincingly demonstrated in this regard. The aim of this trial was to investigate whether combined administration of vitamin B12, folic acid, iron, and erythropoietin could decrease transfusion requirements in extremely low birth weight infants. PATIENTS AND METHODS. In a randomized, controlled trial, extremely low birth weight infants with a birth weight ≤800g and a gestational age ≤32 weeks were randomly assigned to a group receiving combination treatment or a control arm. RESULTS. The treatment increased levels of folate in red blood cells, vitamin B12, ferritin, transferrin receptor levels in plasma, and reticulocyte counts. The proportion of infants requiring no transfusions was lower in the treatment group (38%) as compared with controls (5%). The treatment group and the need for mechanical ventilation were independent predictors of the number of transfusions in multiple regression analysis. Cox regression analysis indicated that combined therapy resulted in a 79% risk reduction for any transfusion. CONCLUSION. Combined treatment with erythropoietin, intravenous iron, folate, and vitamin B12 during the first weeks reduces the need for transfusion in extremely low birth weight infants.

Regulation of Fas (APO-1, CD95) and Fas ligand expression in leukocytes during systemic inflammation in humans.

A potential role of Fas/FasL in sepsis is suggested by recent clinical studies showing that Fas and FasL could serve as markers for severity of sepsis. We sought to determine the effect of endotoxin infusion on expression of Fas and FasL. Healthy volunteers (n = 30) received 2 ng/kg endotoxin i.v. Endotoxin infusion decreased Fas expression on neutrophils and monocytes by 15–20% at 2–4 h in vivo and also in vitro. A rebound increase in Fas (30%) was seen on neutrophils at 24 h, and soluble FasL levels increased by 100% at 24 h. Fas mRNA levels increased 6-fold 4–6 h after endotoxin infusion as measured by real-time polymerase chain reaction. In contrast, FasL-mRNA levels in circulating leukocytes decreased by >80% 2h after lipopolysaccharide infusion. In summary, low-grade endotoxemia induces early down-modulation of Fas on leukocytes, followed by a several-fold increase in Fas-mRNA expression leading to later Fas surface upregulation on neutrophils. The upregulation of Fas expression, Fas mRNA, and later in FasL and sFas levels in endotoxemia replicates the increased fas levels found in septic patients.

Changes in thrombopoiesis and platelet reactivity in extremely low birth weight infants undergoing erythropoietin therapy for treatment of anaemia of prematurity.

Erythropoietin (Epo) is frequently administered to premature infants to stimulate erythropoiesis. There is evidence from studies in animals and healthy adults that Epo also interacts with thrombopoiesis and platelet function. This study investigates the effect of Epo therapy on platelet reactivity, peripheral platelet counts and thiazole orange-positive (TO+) platelets in extremely low birth weight (ELBW) infants. In a randomised-controlled trial, ELBW infants with a birth weight < or =800 g and a gestational age < or =32 weeks were either randomised to a group receiving Epo during the first weeks of life or to a control group. Our results show that thrombin receptor-activating peptide (TRAP-6) -induced expression of P-selectin increased significantly during the first two weeks of Epo treatment. With the exception of week five, the number of TO+ platelets was significantly higher during the first eight weeks in Epo-treated infants compared to controls. The increase of TO+ platelets was not paralleled by an increase in total platelet count. We can conclude that Epo therapy has a short-lasting effect on platelet reactivity toTRAP-6 in ELBW infants during the first two weeks of life. Furthermore, Epo therapy is associated with an increase in the number of TO+ platelets compared to controls.

Regulation of protease-activated receptor 1 (PAR1) on platelets and responsiveness to thrombin receptor activating peptide (TRAP) during systemic inflammation in humans

Thrombin is a coagulation protease that activates platelets, endothelial cells, leukocytes and mesenchymal cells. Thrombin signaling is mediated at least in part by protease-activated receptors (PARs). As little is known about the in vivo regulation of PAR1, this study aimed to characterize the effects of systemic thrombin formation during human endotoxemia on the regulation of PAR1 and the associated responsiveness of human platelets to thrombin receptor activating peptide (TRAP). Endotoxin (2 ng/kg) was infused into 40 healthy men to study the regulation of PAR1 in systemic human inflammation. The SPAN12 antibody was used to determine the in vivo regulation of PAR1. To measure whether modulation of the PAR1 receptor may be associated with altered platelet reactivity, whole blood was stimulated with TRAP ex vivo. Thrombin generation was determined by prothrombin (F(1+2)) fragment. F(1+2) levels increased almost 9-fold from 0.5+/-0.1 nmol/L to 4.5+/-1.9 nmol/L at 4 h (p<0.001). PAR1 decreased by approximately 8% (p<0.001) within 2 h after endotoxin infusion and stayed at those levels until 6 h. Concomitantly, TRAP induced P-selectin expression maximally decreased by 18% (p<0.001) at 6 h. In conclusion, PAR1 expression is down-regulated on platelets during systemic thrombin formation induced by inflammation in humans which results in decreased responsiveness to subsequent stimulation of the PAR1 receptor.

Effects of aspirin and NO-aspirin (NCX 4016) on platelet function and coagulation in human endotoxemia

Acetylsalicylic acid (ASA) prevents thromboembolic events by inhibiting platelet function through blocking of cyclooxygenase type 1 (COX-1). A nitroderivate of ASA, 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)-phenyl ester (NCX 4016) was synthesized, which additionally acts through nitric oxide release. In various in vitro and animal studies NCX 4016 exhibited antithrombotic and anti-platelet properties. We used the standardized model of endotoxin infusion into human volunteers to compare the effects of NCX 4016 and ASA on platelet function and TF-induced coagulation activation. The trial consisted of two parts. In the first part, 10 healthy male volunteers were included in a randomized, open cross-over trial to find a NCX formulation with optimal tolerability and pharmacokinetic data were obtained. The second part was a randomized, double blind placebo controlled clinical trial consisting of 30 healthy male volunteers in three parallel groups (n = 10 per group). Volunteers received either NCX 4016 (800 mg b.i.d.), ASA (425 mg b.i.d.) or placebo for 7 days, before infusion of 2 ng/kg endotoxin on day 8. ASA attenuated the endotoxin-induced platelet plug formation (measured by PFA-100) significantly better than NCX 4016 and placebo (p < 0.004), while there was no difference in soluble P-selectin or VWF-levels. Urine 11-dehydro-thromboxane B2 levels were significantly lower in the ASA and NCX 4016 groups as compared to placebo (p < 0.05). Neither ASA nor NCX 4016 significantly changed prothrombin fragment1 + 2, D-Dimer or tissue factor (TF)-mRNA levels. In summary, NCX 4016 had no effect on VWF release, platelet activation as measured by soluble P-selectin or TF gene expression. NCX 4016, at the dose tested, unlike ASA, had no effect on platelet collagen/epinephrine induced plug formation under high shear rates.

Pharmacokinetics and Pharmacodynamics of the Dual FII/FX Inhibitor BIBT 986 in Endotoxin‐induced Coagulation

BIBT986 is a dual inhibitor of factors Xa and IIa. The aim of this study was to compare with placebo the effect of three doses of BIBT986 on coagulation, platelet activation, and inflammation. This was a prospective, randomized, double‐blind, placebo‐controlled, parallel‐group dose escalation trial in 48 healthy male volunteers. Participants received one of three doses of BIBT986 or placebo intravenously together with a bolus infusion of 2 ng/kg lipopolysaccharide (LPS). BIBT986 dose‐dependently changed global coagulation parameters and in vivo markers of thrombin generation and action: BIBT986 doses, which prolonged activated partial thromboplastin time by 100%, completely suppressed the LPS‐induced increases in prothrombin fragment, thrombin‐antithrombin complexes, and D‐dimer, which were 6.1‐, 14.5, and 3.5‐fold in the placebo group, respectively. BIBT986 did not influence inflammation, fibrinolysis, or platelet activation. Therefore, BIBT986 is a potent anticoagulant in the human endotoxemia model.

Significant Reduction of Catheter-associated Blood Stream Infections in Preterm Neonates After Implementation of a Care Bundle Focusing on Simulation Training of Central Line Insertion.

Background: Central line–associated blood stream infections (CLABSIs) are common problems in neonatal intensive care units (NICUs). Implementation of catheter care bundles has been shown to reduce CLABSI rates. We developed a care bundle aiming at establishing a uniform central line insertion technique and improving teaching practices focusing on simulation-based techniques. The purpose of this study was to assess the impact of this care bundle on CLABSI rates in very low birth weight infants (VLBWI). Methods: In September 2010, a CLABSI prevention bundle was introduced in our NICU, consisting of simulation-based standardization and education of a peripherally inserted central catheter insertion technique. Data of all VLBWI admitted to our NICU during 2010–2012 were analyzed. Diagnosis of CLABSI required a positive blood culture in the presence of a central venous catheter and clinical signs of infection. Results: Five hundred twenty-six VLBWI admitted during the study period were included into the analysis. CLABSI rates decreased significantly from 13.9 in 2010 to 9.5 in 2011 and 4.7 in 2012 (P < 0.0001). This significant reduction was true for the overall population and for subgroups separated by birth weight. Distribution of blood culture pathogens revealed a constant absolute and relative decline of infections with coagulase-negative staphylococci from 2010 (n = 43/50, 86%) to 2012 (n = 12/18, 67%), as opposed by a slight increase of Staphylococcus aureus infections (n = 1/50, 2% in 2010 versus n = 2/18, 11% in 2012). Conclusion: Our data provide evidence of a potential effect of simulation-based training of central line placement in decreasing CLABSI rates in VLBWI and encourage its implementation into care bundles.

Pharmacokinetics and pharmacodynamics of the dual FII/FX inhibitor BIBT 986 in endotoxin-induced coagulation

BIBT986 is a dual inhibitor of factors Xa and IIa. The aim of this study was to compare with placebo the effect of three doses of BIBT986 on coagulation, platelet activation, and inflammation. This was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants received one of three doses of BIBT986 or placebo intravenously together with a bolus infusion of 2 ng/kg lipopolysaccharide (LPS). BIBT986 dose-dependently changed global coagulation parameters and in vivo markers of thrombin generation and action: BIBT986 doses, which prolonged activated partial thromboplastin time by 100%, completely suppressed the LPS-induced increases in prothrombin fragment, thrombin-antithrombin complexes, and D-dimer, which were 6.1-, 14.5, and 3.5-fold in the placebo group, respectively. BIBT986 did not influence inflammation, fibrinolysis, or platelet activation. Therefore, BIBT986 is a potent anticoagulant in the human endotoxemia model.