Vanessa Zambelli

Regulation of leukocyte recruitment by the long pentraxin PTX3

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.

Distinct cardiac and renal effects of ETA receptor antagonist and ACE inhibitor in experimental type 2 diabetes.

Diabetic nephropathy is associated with cardiovascular morbidity. Angiotensin-converting enzyme (ACE) inhibitors provide imperfect renoprotection in advanced type 2 diabetes, and cardiovascular risk remains elevated. Endothelin (ET)-1 has a role in renal and cardiac dysfunction in diabetes. Here, we assessed whether combination therapy with an ACE inhibitor and ET(A) receptor antagonist provided reno- and cardioprotection in rats with overt type 2 diabetes. Four groups of Zucker diabetic fatty (ZDF) rats were treated orally from 4 (when proteinuric) to 8 mo with vehicle, ramipril (1 mg/kg), sitaxsentan (60 mg/kg), and ramipril plus sitaxsentan. Lean rats served as controls. Combined therapy ameliorated proteinuria and glomerulosclerosis mostly as a result of the action of ramipril. Simultaneous blockade of ANG II and ET-1 pathways normalized renal monocyte chemoattractant protein-1 and interstitial inflammation. Cardiomyocyte loss, volume enlargement, and capillary rarefaction were prominent abnormalities of ZDF myocardium. Myocyte volume was reduced by ramipril and sitaxsentan, which also ameliorated heart capillary density. Drug combination restored myocardial structure and reestablished an adequate capillary network in the presence of increased cardiac expression of VEGF/VEGFR-1, and significant reduction of oxidative stress. In conclusion, in type 2 diabetes concomitant blockade of ANG II synthesis and ET-1 biological activity through an ET(A) receptor antagonist led to substantial albeit not complete renoprotection, almost due to the ACE inhibitor. The drug combination also showed cardioprotective properties, which however, were mainly dependent on the contribution of the ET(A) receptor antagonist through the action of VEGF.

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease.

Targeting amyloid-β peptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimers disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation features and to cross in vitro and in vivo the blood-brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aβ (kD=0.6 μM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability=2.5×10(-5) cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD. From the clinical editor: Bi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aβ aggregation/disaggregation as a potential treatment in an Alzheimers model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimers disease.

Alveolar pentraxin 3 as an early marker of microbiologically confirmed pneumonia: a threshold-finding prospective observational study

IntroductionTimely diagnosis of pneumonia in intubated critically ill patients is rather challenging. Pentraxin 3 (PTX3) is an acute-phase mediator produced by various cell types in the lungs. Animal studies have shown that, during pneumonia, PTX3 participates in fine-tuning of inflammation (for example, microbial clearance and recruitment of neutrophils). We previously described an association between alveolar PTX3 and lung infection in a small group of intubated patients. The aim of the present study was to determine a threshold level of alveolar PTX3 with elevated sensitivity and specificity for microbiologically confirmed pneumonia.MethodsWe recruited 82 intubated patients from two intensive care units (San Gerardo Hospital, Monza, Italy, and Massachusetts General Hospital, Boston, MA, USA) undergoing bronchoalveolar lavage (BAL) as per clinical decision. We collected BAL fluid and plasma samples, together with relevant clinical and microbiological data. We assayed PTX3 and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in BAL fluid and PTX3, sTREM-1, C-reactive protein (CRP) and procalcitonin (PCT) in plasma. Two blinded independent physicians reviewed patient data to confirm pneumonia. We determined the PTX3 threshold in BAL fluid for pneumonia and compared it to other biomarkers.ResultsMicrobiologically confirmed pneumonia of bacterial (n =12), viral (n =4) or fungal (n =8) etiology was diagnosed in 24 patients (29%). PTX3 levels in BAL fluid predicted pneumonia with an area under the receiving operator curve of 0.815 (95% CI =0.710 to 0.921, P <0.0001), whereas none of the other biomarkers were effective. In particular, PTX3 levels ≥1 ng/ml in BAL fluid predicted pneumonia in univariate analysis (β =2.784, SE =0.792, P <0.001) with elevated sensitivity (92%), specificity (60%) and negative predictive value (95%). Net reclassification index PTX3 values ≥1 ng/ml in BAL fluid for pneumonia indicated gain in sensitivity and/or specificity vs. all other mediators. These results did not change when we limited our analyses only to confirmed cases of bacterial pneumonia. Moreover, when we considered only the 70 patients who fulfilled the clinical criteria for the diagnosis of pneumonia at BAL fluid sampling, the diagnostic accuracy of PTX levels was confirmed in univariate and ROC curve analysis.ConclusionsIn this hypothesis-generating convenience sample, a PTX3 level ≥1 ng/ml in BAL fluid was discriminative of microbiologically confirmed pneumonia in mechanically ventilated patients.

Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension

AIMS Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. METHODS AND RESULTS PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca(2+) was increased and Ca(2+) release was facilitated. K(+) currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca(2+) content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. CONCLUSION PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

Ibuprofen plus isosorbide dinitrate treatment in the mdx mice ameliorates dystrophic heart structure

BACKGROUND Co-administration of ibuprofen (IBU) and isosorbide dinitrate (ISDN) provides synergistic beneficial effects on dystrophic skeletal muscle. Whether this treatment has also cardioprotective effects in this disease was still unknown. AIMS To evaluate the effects of co-administration of IBU and ISDN (a) on left ventricular (LV) structure and function, and (b) on cardiac inflammatory response and fibrosis in mdx mice. METHODS Three groups of mice were studied: mdx mice treated with IBU (50 mg kg⁻¹)+ISDN (30 mg kg⁻¹) administered daily in the diet, mdx mice that received standard diet without drugs and wild type aged-matched mice. Animals were analysed after 10-11 months of treatment. Structural and functional parameters were evaluated by echocardiography while histological analyses were performed to evaluate inflammatory response, collagen deposition, cardiomyocyte number and area. RESULTS Treatment for 10-11 months with IBU+ISDN preserved LV wall thickness and LV mass. Drug treatment also preserved the total number of cardiomyocytes in the LV and attenuated the increase in cardiomyocyte size, when compared to untreated mdx mice. Moreover, a trend towards a decreased number of inflammatory cells, a reduced LV myocardial interstitial fibrosis and an enhanced global LV function response to stress was observed in treated mdx mice. CONCLUSIONS Treatment for 10-11 months with IBU+ISDN is effective in preventing the alterations in LV morphology of mdx mice while not reaching statistical significance on LV function and cardiac inflammation.

Gold nanoparticles obtained by aqueous digestive ripening: Their application as X-ray contrast agents

A preparative protocol to synthesize large quantities of size-controlled gold nanoparticles (Au NPs), stabilized by CH3O-PEG5000-SH (PEG-SH) in aqueous medium, is reported. The combination of metal vapor synthesis (MVS) technique with digestive ripening process allowed to obtain PEGylated Au NPs with mean core particle size of 3.8nm and hydrodynamic diameters centered at 8.0nm which were effectively used as computed tomography (CT) contrast agents for in vivo experiments on mice. The surface functionalization together with the small hydrodynamic diameters of the engineered Au nanoparticles permitted their efficient renal clearance, still retaining a prolonged blood circulation and a stealth capability.

Angiotensin-(1-7) improves oxygenation, while reducing cellular infiltrate and fibrosis in experimental Acute Respiratory Distress Syndrome

BackgroundThe renin-angiotensin system (RAS) plays a role in the pathogenesis of ARDS, Angiotensin II (Ang-II) contributing to the pathogenesis of inflammation and fibrogenesis. Angiotensin-(1-7) (Ang-(1-7)) may antagonize the effects of Ang-II. This study was aimed at evaluating the potential for Ang-(1-7) to reduce injury, inflammation and fibrosis in an experimental model of ARDS in the acute and late phases.MethodsMale Sprague Dawley rats underwent an instillation of 0.1 M hydrochloric acid (HCl, 2.5 ml/kg) into the right bronchus. In an acute ARDS study, acid-injured rats were subjected to high stretch mechanical ventilation (18 ml/kg) for 5 h and randomized to receive an intravenous infusion of either vehicle (saline), Ang-(1-7) at low dose(0.27 μg/kg/h) (ALD), or high dose (60 μg/kg/h) (AHD) starting simultaneously with injury or 2 h afterwards. Arterial blood gas analysis and bronchoalveolar lavage (BAL) were performed to assess the injury. For the late ARDS study, after HCl instillation rats were randomized to either vehicle or high dose Ang-(1-7) (300 μg/kg/day) infused by mini osmotic pumps for two weeks, and lung hydroxyproline content measured.ResultsIn the acute ARDS study, Ang-(1-7) led to a significant improvement in oxygenation (PaO2/FiO2 : vehicle 359 ± 86; ALD 436 ± 72; AHD 44 442 ± 56; ANOVA p = 0.007) and reduced white blood cells counts (vehicle 4,519 ± 2,234; ALD 2,496 ± 621; AHD 2,744 ± 119/mm3; ANOVA p = 0.004). Only treatment with high dose Ang-(1-7) reduced inflammatory cell numbers in BAL (vehicle 127 ± 34; AHD 96 ± 34/ μl; p = 0.033). Interestingly also delayed administration of Ang-(1-7) was effective in reducing injury. In later ARDS, Ang-(1-7) decreased hydroxyproline content (649 ± 202 and 1,117 ± 297 μg/lung; p < 0.05).ConclusionsAngiotensin-(1-7), decreased the severity of acute lung injury and inflammation induced by combined acid aspiration and high stretch ventilation. Furthermore, continuous infusion of Ang-(1-7) reduced lung fibrosis 2 weeks following acid aspiration injury. These results call for further research on Ang-(1-7) as possible therapy for ARDS.

Efficacy of aminaftone in a rat model of monocrotaline-induced pulmonary hypertension

Pulmonary hypertension is characterized by increased vascular resistances, that could lead to right heart failure and death. Endothelin-1 (ET-1) is a peptide with strong vasoconstrictive and pro-fibrotic properties and is one of the main mediators of pulmonary hypertension. Aminaftone, a synthetic molecule derivative of 4-amynobenzoic acid, down-regulates ET-1 production in vitro by interfering with the transcription of the pre-pro-ET-1 gene. The aim of this study was to test whether the inhibition of ET-1 production by aminaftone attenuates the effects of pulmonary hypertension. Pulmonary hypertension was induced through s.c. injection of 60 mg/kg monocrotaline. The rats were randomly assigned to the following experimental groups: Control; Monocrotaline; Aminaftone 30 mg/kg/day; Aminaftone 150 mg/kg/day. After 5 weeks, mortality was significantly lower in the animals treated with aminaftone at both doses compared to monocrotaline alone. Aminaftone reduced plasma concentration of ET-1 and seemed to reduce right heart hypertrophy and the wall thickness of the pulmonary arteries at the highest dose. Aminaftone may represent a novel treatment strategy of pulmonary hypertension.

Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer’s Aβ peptide

Aggregation of amyloid-β peptide (Aβ) is a key event in the pathogenesis of Alzheimers disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aβ. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aβ oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aβ. PINPs also bound to Aβ with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aβ, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.