Polymicrobial antibiofilm activity of the membranotropic peptide gH625 and its analogue.

This work illustrates a new role for the membranotropic peptide gH625 and its derivative gH625-GCGKKK in impairing formation of polymicrobial biofilms. Mixed biofilms composed of Candida and bacterial species cause frequently infections and failure of medical silicone devices and also show a major drug resistance than single-species biofilms. Inhibition and eradication of biofilms were evaluated by complementary methods: XTT-reduction, and crystal violet staining (CV). Our results indicate that gH625-GCGKKKK, better than the native peptide, strongly inhibited formation of mixed biofilms of clinical isolates of C. tropicalis/S. marcescens and C. tropicalis/S. aureus and reduced the biofilm architecture, interfering with cell adhesion and polymeric matrix, as well as eradicated the long-term polymicrobial biofilms on silicone surface.

Meeting Report EuroG20 Meeting on Cancer-Associated Thrombosis (CAT) Bergamo, Italy 7 April 2016.

The EuroG20 meeting on cancer-associated thrombosis (CAT) convened in Bergamo, Italy on 7 April 2016 to discuss a selection of controversial topics in CAT management. This satellite meeting besides ICTHIC in Bergamo has the objective to propose an European Guidance on CAT in various complex situations where evidence-based guidelines are lacking, driven by eminence-based thoughts of 20 experts and key opinion leaders in thrombosis from EU area and 8 experts from the rest of the world.

Anti-HIV activity of new higher order G-quadruplex aptamers obtained from tetra-end-linked oligonucleotides.

By combining the ability of short G-rich oligodeoxyribonucleotides (ODNs) containing the sequence 5'CGGA3' to form higher order G-quadruplex (G4) complexes with the tetra-end-linked (TEL) concept to produce aptamers targeting the HIV envelope glycoprotein 120 (gp120), three new TEL-ODNs (1-3) having the sequence 5'CGGAGG3' were synthesized with the aim of studying the effect of G4 dimerization on their anti-HIV activity. Furthermore, in order to investigate the effect of the groups at the 5' position, the 5' ends of 1-3 were left uncapped (1) or capped with either the lipophilic dimethoxytrityl (DMT) (2) or the hydrophilic glucosyl-4-phosphate (3) moieties. The here reported results demonstrate that only the DMT-substituted TEL-ODN 2 is effective in protecting human MT-4 cell cultures from HIV infection (76% max protection), notwithstanding all the three new aptamers proved to be capable of forming stable higher order dimeric G4s when annealed in K+-containing buffer, thus suggesting that the recognition of a hydrophobic pocket on the target glycoprotein by the aptamers represents a main structural feature for triggering their anti-HIV activity.

Surface decoration with gH625-membranotropic peptides as a method to escape the endo-lysosomal compartment and reduce nanoparticle toxicity.

The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.

Clinical variables associated with late-onset thrombotic and cardiovascular events, after SARS-CoV-2 infection, in a cohort of patients from the first epidemic wave: an 18-month analysis on the "Surviving-COVID" cohort from Bergamo, Italy.

Importance: Population studies have recorded an increased, unexplained risk of post-acute cardiovascular and thrombotic events, up to 1 year after acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To search for clinical variables and biomarkers associated with late post-acute thrombotic and cardiovascular events after SARS-CoV-2 infection. Design: Retrospective cohort study. Setting: Third-level referral hospital in Bergamo (Italy). Participants: Analysis of an existing database of adult patients, who received care for SARS-CoV-2 infection at our institution between 20 February and 30 September 2020, followed up on a single date ("entry date") at 3-6 months. Exposure: Initial infection by SARS-CoV-2. Main outcomes and measures: Primary outcome: occurrence, in the 18 months after entry date, of a composite endpoint, defined by the International Classification of Diseases-9th edition (ICD-9) codes for at least one of: cerebral/cardiac ischemia, venous/arterial thrombosis (any site), pulmonary embolism, cardiac arrhythmia, heart failure. Measures (as recorded on entry date): history of initial infection, symptoms, current medications, pulmonary function test, blood tests results, and semi-quantitative radiographic lung damage (BRIXIA score). Individual clinical data were matched to hospitalizations, voluntary vaccination against SARS-CoV-2 (according to regulations and product availability), and documented reinfections in the following 18 months, as recorded in the provincial Health Authority database. A multivariable Cox proportional hazard model (including vaccine doses as a time-dependent variable) was fitted, adjusting for potential confounders. We report associations as hazard ratios (HR) and 95% confidence intervals (CI). Results: Among 1,515 patients (948 men, 62.6%, median age 59; interquartile range: 50-69), we identified 84 endpoint events, occurring to 75 patients (5%): 30 arterial thromboses, 11 venous thromboses, 28 arrhythmic and 24 heart failure events. From a multivariable Cox model, we found the following significant associations with the outcome: previous occurrence of any outcome event, in the 18 months before infection (HR: 2.38; 95% CI: 1.23-4.62); BRIXIA score ≥ 3 (HR: 2.43; 95% CI: 1.30-4.55); neutrophils-to-lymphocytes ratio ≥ 3.3 (HR: 2.60; 95% CI: 1.43-4.72), and estimated glomerular filtration rate < 45 ml/min/1.73 m2 (HR: 3.84; 95% CI: 1.49-9.91). Conclusions and relevance: We identified four clinical variables, associated with the occurrence of post-acute thrombotic and cardiovascular events, after SARS-CoV-2 infection. Further research is needed, to confirm these results.

The impact of perioperative transfusion of blood products on survival after pediatric liver transplantation.

Intraoperative transfusion of red blood cells (RBC) is associated with adverse outcome after LT in adult patients. This relationship in pediatric patients has not been studied in depth, and its analysis is the scope of this study. Forty-one variables associated with outcome, including blood product transfusions, were studied in a cohort of 243 pediatric patients undergoing a cadaveric LT between 2002 and 2009 at the General Hospital of Bergamo. Multivariate stepwise Cox proportional hazards models were adopted with adjustment by propensity scores to minimize factors associated with the use of blood products. Median age at transplant was 1.37 yr. In uni- and multivariate analyses, perioperative transfusion of FFP and RBC was an independent risk factor for predicting one-yr patient and graft survival. The effect on one-yr survival was dose-related with a hazard ratio of 3.15 for three or more units of RBC (p = 0.033) and 3.35 for three or more units of FFP (p = 0.021) when compared with 1 or no units transfused. The negative impact of RBC and FFP transfusion was confirmed by propensity score-adjusted analysis. These findings may have important implications for transfusion practice in the LT pediatric recipients.

Lombardy diagnostic and therapeutic network of thrombotic microangiopathy.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA) requiring urgent treatment. Standardization of its diagnosis and optimal management is challenging. This study aimed to evaluate the role of centralized, rapid testing of ADAMTS13 in patients experiencing acute TMAs requiring plasma-exchange (PEX) and to estimate the incidence of TTP in a large Italian Region. METHODS: We perfomed a cohort study in the frame of the project "Set-up of a Lombardy network for the study and treatment of patients undergoing apheresis", including 11 transfusion centers in the Region. Consecutive patients referred from 2014 to 2016 with acute TMAs requiring PEX were enrolled. Centralized ADAMTS13 activity testing was performed at the Milan Hemophilia and Thrombosis Center within 24 h. RESULTS: Forty-three TMA patients (44 events) were enrolled, of whom 35 (81%) had severe ADAMTS13 deficiency. Patients with severe ADAMTS13 deficiency were younger, mainly women, with a higher prevalence of autoimmune disorders and a lower prevalence of cancer. Clinical and laboratory characteristics of patients with and without severe ADAMTS13 deficiency largely overlapped, with a lower platelet count being the only baseline marker that significantly differed between the two patient groups (ADAMTS13 activity < 10% vs ≥ 10%: median difference of -27 × 109/l, 95% CI - 37 to - 3). PEX treatment was initiated in all patients, but soon discontinued in cases without severe ADAMTS13 deficiency. In this group, the mortality rate was higher and no episode exacerbations or relapses within 6 months occured. The estimated average annual incidence of acute acquired TTP events was 1.17 [0.78-1.55] per million people. CONCLUSIONS: Severe ADAMTS13 deficiency distinguished two groups of patients with largely overlapping clinical features but different treatment and disease course. This study provides a feasible model implemented in a large Italian region for the practical clinical approach to TMAs and underlines the importance of urgent ADAMTS13 activity testing for an accurate differential diagnosis and therapeutic approach.

Acquired and inherited risk factors for developing venous thromboembolism in cancer patients receiving adjuvant chemotherapy: a prospective trial.

BACKGROUND: Acquired and inherited risk factors for venous thromboembolism (VTE) and the incidence of symptomatic VTE were investigated in patients on adjuvant chemotherapy for breast or gastrointestinal cancer (GI). PATIENTS AND METHODS: In a prospective observational study (January 2003 and February 2006), 199 GI (82 women/117 men; age range, 26-84 years) and 182 breast (180 women/2 men; age range, 29-85 years) cancer patients were enrolled and followed-up for symptomatic VTE during adjuvant chemotherapy. The effect of acquired (i.e. age, chemotherapy, tumour histotype, history of thrombosis, body mass index and smoking) and inherited risk factors [i.e. antithrombin, protein C (PC), protein S, homocysteine, activated PC resistance, factor V Leiden (FVL) and prothrombin (PT) mutations) was prospectively evaluated. RESULTS: Overall, 30 VTE events (7.87%) were recorded: 28 (7.35%) during treatment and 2 (0.52%) during the subsequent follow-up. Among all the 381 cancer patients, FVL was detected in 14 cases (3.67%) and PT mutation in 10 cases (2.62%). Multivariate analysis showed a significant association between the development of VTE and both thrombocytosis [hazard ratio (HR) 1.65; 95% confidence interval (CI), 1.04-2.637, P <0.0341] and a prior episode of thrombosis (HR 7.6; 95% CI, 1.77-33.1, P <0.006). FVL and PT mutations were not associated with the risk for VTE. CONCLUSION: The present data indicate thrombocytosis and history of thrombosis as risk factors for development of a thrombotic event during adjuvant chemotherapy in patients with malignant diseases.

Endogenous and artificial miRNAs explore a rich variety of conformations: a potential relationship between secondary structure and biological functionality.

Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.

Platelet thromboxane synthetase inhibitors with low doses of aspirin: possible resolution of the "aspirin dilemma".

Selective pharmacological inhibition of thromboxane A2 synthesis did not prevent arachidonate-induced aggregation of human platelets in vitro. Prevention was instead achieved by a combination of thromboxane A2 inhibitors with low concentrations of aspirin. The latter partially reduced the proaggregatory cyclooxygenase products that accumulated when thromboxane A2 synthesis was blocked. The aspirin concentrations did not affect per se either platelet aggregation or prostacyclin synthesis in cultured human endothelial cells. The combination of thromboxane synthetase inhibitors with low doses of aspirin may offer greater antithrombotic potential than either drug alone.

Antithrombotic medication in cancer-associated thrombocytopenia: Current evidence and knowledge gaps.

In cancer patients, antithrombotic medications (i.e. anticoagulation or antiplatelet therapy) are frequently prescribed for prior or new indications such as venous thromboembolism or stoke prevention in atrial fibrillation. Balancing the risks of bleeding and thrombosis during periods of thrombocytopenia represents a significant challenge. Management is informed mainly by expert opinion and several recent retrospective studies on venous thromboembolism. The main management options include no change, temporarily withholding antithrombotic therapy, reducing dose, changing the regimen, and increasing the platelet transfusion threshold. Important recent advances in knowledge include the prognostic importance and apparent safety of aspirin in acute myocardial infarction and thrombocytopenia and data suggesting a low risk of recurrent venous thromboembolism in autologous stem cell transplantation patients who had anticoagulation withheld. This paper will review the literature on antithrombotic medication in thrombocytopenic patients with cancer. The significant knowledge gaps will be summarized and considerations for practice and research will be provided.

Comparison Between Folic Acid and gH625 Peptide-Based Functionalization of Fe3O4 Magnetic Nanoparticles for Enhanced Cell Internalization.

A versatile synthetic route based on magnetic Fe3O4 nanoparticle (MNP) prefunctionalization with a phosphonic acid monolayer has been used to covalently bind the gH625 peptide on the nanoparticle surface. gH625 is a membranotropic peptide capable of easily crossing the membranes of various cells including the typical human blood-brain barrier components. A similar synthetic route was used to prepare another class of MNPs having a functional coating based on PEG, rhodamine, and folic acid, a well-known target molecule, to compare the performance of the two cell-penetrating systems (i.e., gH625 and folic acid). Our results demonstrate that the uptake of gH625-decorated MNPs in immortalized human brain microvascular endothelial cells after 24 h is more evident compared to folic acid-functionalized MNPs as evidenced by confocal laser scanning microscopy. On the other hand, both functionalized systems proved capable of being internalized in a brain tumor cell line (i.e., glioblastoma A-172). These findings indicate that the functionalization of MNPs with gH625 improves their endothelial cell internalization, suggesting a viable strategy in designing functional nanostructures capable of first crossing the BBB and, then, of reaching specific tumor brain cells.

Blood oxidative status and selectins plasma levels in healthy donors receiving granulocyte-colony stimulating factor.

Recombinant human G-CSF (rHuG-CSF) is used for hematopoietic progenitor cells (HPC) mobilization and collection. Activation of polymorphonuclear leukocytes (PMN) is present during rHuG-CSF treatment and is associated with endothelial cell dysfunction and hypercoagulation. We evaluated whether PMN activation by rHuG-CSF may alter the blood oxidative status and subsequently affect the vascular cell function. Fourteen healthy individuals received rHuG-CSF for HPC harvesting. Blood was drawn before starting rHuG-CSF (T0), on the last day of rHuG-CSF (T1) and 1 week after stopping rHuG-CSF (T2). Levels of CD11b, myeloperoxidase (MPO), hydroperoxides, nitric oxide (NO), and soluble endothelium (sES), leukocyte (sLS), and platelet (sPS) selectins were measured. During rHuG-CSF, CD11b, MPO and hydroperoxides significantly increased, while NO levels significantly decreased, compared with T0. At T2 all these markers returned to baseline values. Significant increments of all selectins were observed during rHuG-CSF. At T2 sES and sEP significantly decreased back to pre-treatment values, whereas sLS remained significantly high. These data show that rHuG-CSF induces a transient inflammatory status characterized by circulating activated PMN, which release reactive oxygen species and intracellular proteases, promoting the onset of an abnormal oxidative status. This process may modify the hemostatic balance towards a pro-thrombotic state.

Identification of the new HLA-DRB1*14:186 allele in an Italian bone marrow donor.

HLA-DRB1*14:186 differs from DRB1*14:58 by a non-synonymous mutation at nucleotide 227 in exon 2.

External validation of the DASH prediction rule: a retrospective cohort study.

Essentials Predicting recurrences may guide therapy after unprovoked venous thromboembolism (VTE). We evaluated the DASH score in 827 patients with unprovoked VTE to verify prediction accuracy. A DASH score ≤ 1 had a cumulative recurrence risk at 1 year of 3.6%, as predicted by the model. The DASH score performed better in younger (< 65 years old) subjects. SUMMARY: Background The DASH prediction model has been proposed as a guide to identify patients at low risk of recurrence of venous thromboembolism (VTE), but has never been validated in an independent cohort. Aims To validate the calibration and discrimination of the DASH prediction model, and to evaluate the DASH score in a predefined patient subgroup aged > 65 years. Methods Patients with a proximal unprovoked deep vein thrombosis (DVT) or pulmonary embolism (PE) who received a full course of vitamin K antagonist or direct oral anticoagulant (> 3 months) and had D-dimer measured after treatment withdrawal were eligible. The DASH score was computed on the basis of the D-dimer level after therapy withdrawal and personal characteristics at the time of the event. Recurrent VTE events were symptomatic proximal or distal DVT/PE, and were analyzed with a time-dependent analysis. Observed 12-month and 24-month recurrence rates were compared with recurrence rates predicted by the DASH model. Results We analyzed a total of 827 patients, of whom 100 (12.1%) had an objectively documented recurrence. As compared with the original DASH cohort, there was a greater proportion of subjects with a 'low-risk' (≤ 1) DASH score (66.3% versus 51.6%, P < 0.001). The slope of the observed versus expected cumulative incidence at 2 years was 0.71 (95% confidence interval 0.51-1.45). The c-statistic was lower for subjects aged > 65 years (0.54) than for younger subjects (0.72). Conclusions These results confirm the validity of DASH prediction model, particularly in young subjects. The recurrence risk in elderly patients (> 65 years) was, however, > 5% even in those with the lowest DASH scores.

Venous thromboembolism and cancer: guidelines of the Italian Association of Medical Oncology (AIOM).

Thromboembolic complications represent one of the most important cause of morbidity and mortality in cancer patients. Although several data have been published demonstrating the strong association between cancer and venous thromboembolism (VTE), there is poor perception, among oncologists, of the level of risk of thrombosis and of relevance of managing VTE in these patients. The Associazione Italiana di Oncologia Medica (AIOM) has provided some recommendations to direct clinical practice according to evidence-based data concerning cancer and VTE. In fact, we conducted an extensive literature review (1996-2005) to produce evidence-based recommendations to improve perceptions of the magnitude of this risk among Italian medical and surgical oncologists and alert on the new approaches to prophylaxis and treatment of VTE in cancer patients. Levels of evidence are given according to a five-point rating system, and similarly for each key recommendation a five-point rating system suggests if the evidence is strong and indicate that the benefits do, or do not, outweigh risks and burden.