Davide Piloni

Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction.

AIMS Chronic lung allograft dysfunction represents the main cause of death after lung transplantation, and so far there is no effective therapy. Mesenchymal cells (MCs) are primarily responsible for fibrous obliteration of small airways typical of chronic lung allograft dysfunction. Here, we engineered gold nanoparticles containing a drug in the hydrophobic section to inhibit MCs, and exposing on the outer hydrophilic surface a monoclonal antibody targeting a MC-specific marker (half-chain gold nanoparticles with everolimus). MATERIALS & METHODS Half-chain gold nanoparticles with everolimus have been synthesized and incubated with MCs to evaluate the effect on proliferation and apoptosis. RESULTS & DISCUSSION Drug-loaded gold nanoparticles coated with the specific antibody were able to inhibit proliferation and induce apoptosis without stimulating an inflammatory response, as assessed by in vitro experiments. CONCLUSION These findings demonstrate the effectiveness of our nanoparticles in inhibiting MCs and open new perspectives for a local treatment of chronic lung allograft dysfunction.

Interleukin-17 receptor polymorphism predisposes to primary graft dysfunction after lung transplantation

BACKGROUND Primary graft dysfunction (PGD), with an incidence of 11% to 57%, is a major cause of morbidity and mortality within the first 30 days after lung transplantation (LTx). In this study, we postulate that recipient genetic variants in interleukin-17 and -23 receptor genes (IL-17R and IL-23R, respectively) may predispose LTx recipients to an increased risk for developing PGD. METHODS Seven genetic variants of IL-17R and IL-23R were successfully genotyped in 431 lung transplant recipients. Our primary end-point was PGD and secondary end-points were time to extubation, intensive care unit (ICU) stay, bronchoalveolar lavage neutrophilia and serum C-reactive protein. RESULTS The AA genotype of the rs882643 genetic variant of IL-17R was associated with higher PGD grades at 0 hour (adjusted p = 0.042), 12 hours (adjusted p = 0.013) and 48 hours (adjusted p = 0.0092) after LTx. The GG genotype of the rs2241049 genetic variant of IL-17R was associated with higher PGD grades at 48 hours (adjusted p = 0.0067) after LTx. For both genetic variants, no association was found with extubation time, ICU stay, post-operative BAL neutrophilia, serum CRP, chronic lung allograft dysfunction (CLAD) or graft loss. CONCLUSION Both genetic variants of IL-17R (rs882643 and rs2241049) were associated with PGD. This confirms a genetic predisposition toward PGD and suggests a role of IL-17 in driving neutrophilia in PGD.

The combination of sorafenib and everolimus shows antitumor activity in preclinical models of malignant pleural mesothelioma

BackgroundMalignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify ‘druggable’ pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus.MethodsWe planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in ‘hot spot’ regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA).ResultsPhosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice.ConclusionsERM and mTOR pathways are activated in MPM and ‘druggable’ by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM.

The Role of a Panel of Pro-Fibrogenic miRs in Fibrotic Lung Disorders

Rationale: Pulmonary idiopathic fibrosis (IPF), Cryptogenetic organizing pneumonia (COP) and bronchiolitis obliterans syndrome (BOS) are rare pulmonary disorders, linked by the presence of fibrotic lesions. In our previous work (Di Carlo, 2016) on BOS we computationally identified a panel of candidate miRNAs and demonstrated by in situ hybridization analysis (ISH) and qRT-PCR, a dysregulation of two highly ranked miRNAs, miR-21 and miR-34a;ISH confirmed abnormal miR-21 and miR-34a expression in BOS lesions; other miRNAs where indicated as potential candidates in BOS by computational analysis. Aim We extended our previous work by analyzing the expression of miR-21, miR-34a and three other highly ranked miRNAs (miR-145, miR-146b-5p and miR-381) in BOS and other lung diseases associated with fibroblast activation/proliferation and collagen deposition. Identifying a specific profile of dysregulated miRNAs could provide useful diagnostic markers and potential therapeutic target. Methods :We evaluated miRNAs expression profile by ISH and RT-PCR quantification in a series of formalin-fixed and paraffin-embedded lung samples obtained from patients with IPF (n. 8), OP (n. 8), BOS (n. 12) and normal lung from organ donors. Results In BOS, COP and IPF/UIP miR-21 and miR-145 were expressed in fibroblasts of BO lesions, OP plugs and in fibroblast foci respectively, and in reactive alveolar epithelia; miR-146b expression correlated to the amount of inflammatory cell infiltrates and epithelial activation in all cases, while a weak expression was evident in OP and IPF/UIP lesions. miR-34a overexpression was associated with the activation of alveolar epithelia and to a lesser extent with fibroblast lesions in OP. miR-381 showed a weak expression in all diseases, and was localized especially in inflammatory cells. ISH data have been confirmed by qRTPCR analysis obtained on same samples. Conclusions: miR-21, miR-145 and miR-146b are over-expressed in fibroblasts in all the cases analyzed, but their expression is not disease-specific, although some differences are observed in different diseases. This finding underlies their role in non-specific fibrotic lung processes.ISH complements the results of qPCR, allows the precise cellular localization of miR expression, and improves correlations with cell-specific pathways

Clinical utility of CD4 + function assessment (ViraCor-IBT ImmuKnow test) in lung recipients

The ImmuKnow assay measures cell-mediated immunity, quantifying ATP production from peripheral blood CD4+T-cells in solid-organ transplant patients who undergo immunosuppressive therapy. We aimed to measure functional immunity in lung transplant recipients and correlate Immuknow values with immunosuppression levels, presence of chronic lung allograft dysfunction (CLAD) and infections. We evaluated 61 lung recipients who underwent follow-up for lung transplantation between 2010 and 2014. Rejection and infection were retrospectively analyzed. The association between over-immunosuppression and a number of predictors was assessed by means of univariate and multivariate logistic regression models. 71 out of 127 samples (56%) showed an over-immunosuppression with an ImmuKnow assay mean level of 112.92ng/ml (SD±58.2), vs. 406.14ng/ml (SD±167.7) of the rest of our cohort. In the over-immunosuppression group we found 51 episodes of infection (71%) (OR 2.754, 95% CI 1.40-5.39; P-value 0.003). In the other group, only 25 samples (44%) were taken during an infectious episode. The mean absolute ATP level was significantly different between patients with or without infection (202.38±139.06ng/ml vs. 315.51±221.60ng/ml; P<0.001). RAS (Restrictive allograft syndrome) was associated to low ImmuKnow level (P<0.001). These results were confirmed by the multivariate analysis. The ImmuKnow assay levels were significantly lower in infected lung transplant recipients compared with non-infected recipients and in RAS patients.

Effect of induction therapy on peripheral blood lymphocytes after lung transplantation: A multicenter international study

Abstract Introduction Lymphocytes, which are targeted by immunosuppressive therapies, may be influenced by induction and recipient characteristics. The objective of this study was to evaluate the influence of induction therapy on lymphocyte kinetics after lung transplantation (LTx) according to the recipient characteristics and allograft outcomes. Methods We retrospectively collected total lymphocyte counts from peripheral blood, which was monitored before and after transplantation (days 7, 14, 30, 90, 180, 365 and 730) in patients from 3 different lung transplant centers in Europe who encountered different induction strategies (no induction, anti-thymocyte globulins and basiliximab). Results A total of 164 recipients were included: 50 patients who did not receive induction therapy and 114 patients who received induction therapy (57 with anti-thymocyte globulins and 57 with basiliximab). The pre-transplant lymphocyte levels and induction therapy were associated with higher lymphocyte differences between pre-transplantation and day 7 (p  Conclusion The blood lymphocyte count after LTx is associated with the pre-transplant lymphocyte level, age, weight and induction therapy and predicts one-year mortality.

Bioengineered gold nanoparticles targeted to mesenchymal cells from patients with bronchiolitis obliterans syndrome does not rise the inflammatory response and can be safely inhaled by rodents

Abstract The use of gold nanoparticles (GNPs) as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long-term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favors epithelial–mesenchymal transition and activation of local mesenchymal cells (MCs) leading to airway fibro-obliteration. In a previous work, we engineered GNPs loaded with the mammalian target of rapamycin inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary MCs isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these GNPs (GNP-HCe) were able to specifically inhibit primary MCs without affecting the bronchial epithelial cell. In the present work, we investigated the effect of these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibrogenesis. In addition, we administered GNP-HCe by the inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. The present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are non-toxic.