This study addresses a joint nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy approach to provide a platform for dynamic assessment of kidney viability and metabolism. On porcine kidney models, ROS production, oxidative damage kinetics, and metabolic changes occurring both during the period between organ retrieval and implantation and after kidney graft were examined. The 1H-NMR metabolic profile—valine, alanine, acetate, trimetylamine-N-oxide, glutathione, lactate, and the EPR oxidative stress—resulting from ischemia/reperfusion injury after preservation (8 h) by static cold storage (SCS) and ex vivo machine perfusion (HMP) methods were monitored. The functional recovery after transplantation (14 days) was evaluated by serum creatinine (SCr), oxidative stress (ROS), and damage (thiobarbituric-acid-reactive substances and protein carbonyl enzymatic) assessments. At 8 h of preservation storage, a significantly (p < 0.0001) higher ROS production was measured in the SCS vs. HMP group. Significantly higher concentration data (p < 0.05–0.0001) in HMP vs. SCS for all the monitored metabolites were found as well. The HMP group showed a better function recovery. The comparison of the areas under the SCr curves (AUC) returned a significantly smaller (−12.5 %) AUC in the HMP vs. SCS. EPR-ROS concentration (μmol·g−1) from bioptic kidney tissue samples were significantly lower in HMP vs. SCS. The same result was found for the NMR monitored metabolites: lactate: −59.76%, alanine: −43.17%; valine: −58.56%; and TMAO: −77.96%. No changes were observed in either group under light microscopy. In conclusion, a better and more rapid normalization of oxidative stress and functional recovery after transplantation were observed by HMP utilization.

Bronchiolitis Obliterans Syndrome seriously reduces long-term survival of lung transplanted patients. Up to now there is no effective therapy once BOS is established. Nanomedicine introduces the possibility to administer drugs locally into lungs increasing drug accumulation in alveola reducing side effects. Imatinib was loaded in gold nanoparticles (GNP) functionalized with antibody against CD44 (GNP-HCIm). Lung fibroblasts (LFs) were derived from bronchoalveolar lavage of BOS patients. GNP-HCIm cytotoxicity was evaluated by MTT assay, apoptosis/necrosis and phosphorylated-cAbl (cAbl-p). Heterotopic tracheal transplantation (HTT) mouse model was used to evaluate the effect of local GNP-HCIm administration by Alzet pump. GNP-HCIm decreased LFs viability compared to Imatinib (44.4 ± 1.8% vs. 91.8 ± 3.2%, p < 0.001), inducing higher apoptosis (22.68 ± 4.3% vs. 6.43 ± 0.29; p < 0.001) and necrosis (18.65 ± 5.19%; p < 0.01). GNP-HCIm reduced cAbl-p (0.41 GNP-HCIm, 0.24 Imatinib vs. to control; p < 0.001). GNP-HCIm in HTT mouse model by Alzet pump significantly reduced tracheal lumen obliteration ( p < 0.05), decreasing apoptosis ( p < 0.05) and TGF-β-positive signal ( p < 0.05) in surrounding tissue. GNP-HCIm treatment significantly reduced lymphocytic and neutrophil infiltration and mast cells degranulation ( p < 0.05). Encapsulation of Imatinib into targeted nanoparticles could be considered a new option to inhibit the onset of allograft rejection acting on BOS specific features.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. Methods: Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n=28) and to the Intermediate Medicine Ward (IMW) (n=5). We analyze the differential cell count, ultrastructure of cells and Interleukin(IL)6, 8 and 10 levels.Results: ICU patients showed a marked increase in neutrophils (1.24 x 105 ml-1 , 0.85-2.07), lower lymphocyte (0.97 x 105 ml-1, 0.024-0.34) and macrophages fractions (0.43 x 105 ml-1, 0.34-1.62) compared to IMW patients (0.095 x 105 ml-1, 0.05-0.73; 0.47 x 105 ml-1, 0.28-1.01 and 2.14 x 105 ml-1, 1.17-3.01, respectively) (p<0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p<0.01, IL8 p<0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (p<0.1) or antivirals (p<0.05). Conclusions: Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.

Purpose Bronchiolitis Obliterans Syndrome (BOS), major clinical phenotype of chronic lung allograft dysfunction (CLAD), is responsible of poor long-term survival after lung transplantation (LTx). Once BOS starts can at best be halted for some months and no pharmacological treatment is able to revert it. Main actors of BOS are aberrantly activated mesenchymal cells (MCs), which proliferate and obliterate the small airways. Based on previous encouraging results with CD44 coated-drug loaded gold nanoparticles (Cova, 2014 and 2017) aiming to engineer more biocompatible nanocarriers to be delivered by inhalation route, we designed hyaluronic acid (HA)- decorated liposomes (LIP-HA) to be loaded with an antiproliferative drug, targeting CD44 receptor on BOS patient-derived MCs Methods We preliminarily evaluated the efficiency of liposomes functionalized with HA of different molecular weight (MW 4800, 14800 kDa), possibly associated to different targeting efficiency and pro-inflammatory activity. Results Using confocal microscopy and flow cytometry, we assessed that LIP decorated with higher MW HA were able to be internalized by MCs more (89 ± 15%) than LIP carrying HA with lower MW (45 ± 7.8%). In order to confirm that our nanovehicles are specific only for CD44-expressing cells, we incubated CD44-negative cell line (16HBE) with both MW HA-liposomes which were poorly internalized (confocal microscopy). We also studied the influence of LIP-HA on cytokine release from macrophages. We observed that 14800 kDa LIP-HA did not alter the release of TNF-α, IL-1β, VEGF and TGF-β, in contrast to 4800 kDa LIP-HA that exerted a 2-fold increase of the release of TGF-β respect to untreated cells (p<0.05). Conclusion In conclusion 14.800- HA decorated liposomes are more efficient in entering primary BOS MCs and do not induce macrophage activation, thus they are promising biocompatible nanovectors for local targeted drug delivery. The efficacy of drug loaded 14.800 HA decorated liposomes is now under investigation.

Purpose Malignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth. Methods MPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry). Results GNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization. Conclusion GNP-HCPe treatment displays in vitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy.

Background: The role of CD19+CD24(high)CD38(high) B-regulatory cells in solid-organ Transplant (Tx) in acceptance are still scarce. In previous studies on kidney transplant recipients may suggest a protective role of this cell subtype in graft tolerance and the existence of a cross talk between B-and T-regulatory clones. In lung transplantation, the role of B-regulatory cells has never been investigated. In a murine tracheal transplantation model, this subset seems able to prevent tracheal obliteration when in combination with rapamycin. Aim of this study is to analyze peripheral CD19+CD24(high)CD38(high) B-reg cells counts in a cohort of lung recipients, their association with several clinical and pharmacological variables and their possible association with T regulatory cell. Methods: From Jan 2009 to Dec 2014, 117 lung Tx recipients were submitted to an immunological follow up I-FU(median: 108.7 months (6.7-310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD19+CD24(high)CD38(high) B-cells (globally 1106 determinations). We tested the association between B-reg and relevant variables by linear or regression models for repeated measures, adjusting for time from Tx. Results: Among all variables analyzed at multivariate analysis: chronic rejection (OR - 0.19, p = .039), use of Mycophenolate (OR - 0.38, p < .001) and the presence of a concomitant pulmonary infection of S. aureus (OR 0.66, p = .002) and A. fumigatus (OR 0.50, p = .009) were significantly associated to B-reg cell. No significant correlation between CD19+CD24(high)CD38(high) B-reg cells and T-reg cells counts was found in our cohort. Conclusions: Our present data highlight, for the first time, that this cell subset might participate in long-term lung graft acceptance mechanisms.

Purpose The use of gold nanoparticles (GNPs) as local targeted drug delivery system are a promising issue for several orphan diseases. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction limiting long term survival after lung transplant, and occurs also as chronic pulmonary involvement in the context of GVHD. To now treatment strategies in BOS are scarce and poorly effective. In the present work, we investigated the effect of GNP decorated with the half chain of monoclonal antibody against CD44 (GNP-HC), surface receptor overexpressed by primary BOS derived mesenchymal cells (MCs) that cause the partial or total occlusion of alveolar tract. We loaded GNP-HC with imatinib (GNP-HCim), an cAbl inhibitor used in the management of chronic GVHD. Methods In vitro experiments were performed on MCs derived from BOS patients evaluating cell cytotoxicity after incubation with GNP-HC, GNP-HCim and Imatinib alone. We further performed a mouse heterotopic trachea transplantation model modified by the subcutaneous placement of Alzet Model 2004 miniosmotic pumps used to deliver into the trachea different solutions at a constant rate for 28 days. This was performed in order to assess the efficacy of the local intratracheal administration of our nanovectors. Results Thanks to MTT and apoptosis assay we observed that GNP-HCim decrease MCs viability more than GNP-HC and Imatinib alone, due to an induction of apoptosis (50 ± 0.5 % for GNP-HCim in contrast to GNP-HC 40 ± 0.35 % and Imatinib alone 30 ± 1.2 %). Moreover, animal model data confirmed the in vitro results: GNP-HCim significantly reduced the percentage of tracheal obliterative area (12.99 ± 2.5%) respect to GNP-HC (46.79± 5.7%) and vehicle treated mice (30.92 ± 5.8%). Conclusion We conclude that this approach by newly developed nano-based strategies for BOS treatment and especially targeted imatinib gold nanoparticles are promising candidates for further development. The use of gold nanoparticles (GNPs) as local targeted drug delivery system are a promising issue for several orphan diseases. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction limiting long term survival after lung transplant, and occurs also as chronic pulmonary involvement in the context of GVHD. To now treatment strategies in BOS are scarce and poorly effective. In the present work, we investigated the effect of GNP decorated with the half chain of monoclonal antibody against CD44 (GNP-HC), surface receptor overexpressed by primary BOS derived mesenchymal cells (MCs) that cause the partial or total occlusion of alveolar tract. We loaded GNP-HC with imatinib (GNP-HCim), an cAbl inhibitor used in the management of chronic GVHD. In vitro experiments were performed on MCs derived from BOS patients evaluating cell cytotoxicity after incubation with GNP-HC, GNP-HCim and Imatinib alone. We further performed a mouse heterotopic trachea transplantation model modified by the subcutaneous placement of Alzet Model 2004 miniosmotic pumps used to deliver into the trachea different solutions at a constant rate for 28 days. This was performed in order to assess the efficacy of the local intratracheal administration of our nanovectors. Thanks to MTT and apoptosis assay we observed that GNP-HCim decrease MCs viability more than GNP-HC and Imatinib alone, due to an induction of apoptosis (50 ± 0.5 % for GNP-HCim in contrast to GNP-HC 40 ± 0.35 % and Imatinib alone 30 ± 1.2 %). Moreover, animal model data confirmed the in vitro results: GNP-HCim significantly reduced the percentage of tracheal obliterative area (12.99 ± 2.5%) respect to GNP-HC (46.79± 5.7%) and vehicle treated mice (30.92 ± 5.8%). We conclude that this approach by newly developed nano-based strategies for BOS treatment and especially targeted imatinib gold nanoparticles are promising candidates for further development.

Gold nanoparticles (GNPs) can be exploited for local and targeted treatment for poorly prognosis diseases. Bronchiolitis Obliterans Syndrome (BOS) is the main cause of medium-term failure of lung transplantation and characterized by fibroobliteration of small airways due to uncontrolled proliferation of mesenchymal cells (MCs). Since CD44 receptor is overexpressed by BOS MCs, we developed GNPs decorated with the half chain of moAb against CD44 (GNP-HC). We aimed firslty to assess the in vitro activity of GNP-HC loaded with imatinib (GNP-HCim), a cAbl inhibitor already used in the treatment of chronic pulmonary GVHD. Furthermore we aimed to evaluate efficacy of local delivery of these carrier in heterotopic tracheal transplantation model in mice. For in vitro experiments, we evaluated cytotoxicity of GNP-HC, GNP-HCim and imatinib alone in BOS-derived MCs. Moreover, we performed in vivo experiments in mouse heterotopic trachea transplantation, with continuous intratracheal administration of GNP or vehicle by Alzet Model 2004 miniosmotic pumps, in order to assess their efficacy. GNP-HCim treated MCs showed significantly reduced viability than GNP-HC and imatinib alone, associated to induction of apoptosis (50 ± 0.5 % for GNP-HCim vs. GNP-HC 40 ± 0.35 % and Imatinib alone 30 ± 1.2 %). These results were strengthened by animal model, in which the percentage of tracheal obliterated area was significantly reduced by GNP-HCim (12.99 ± 2.5%) respect to GNP-HC (46.79± 5.7%) and vehicle treated tracheas (30.92 ± 5.8%). GNPs represent a valid vehicle for local and selective drug administration avoiding systemic toxicity.

Interstitial lung involvement in Systemic Sclerosis (SSc-ILD) is a complication with high morbidity and mortality. Specifically, engineered gold nanoparticles (GNPs) are proposed as targeted delivery system increasing efficacy of drugs with antifibrotic effect, such as tyrosine kinases. We aimed to test in vitro and in vivo the activity of targeted Imatinib (Im)-loaded GNP on SSc-ILD patients derived cells and in experimental model of lung fibrosis. GNPs functionalized with anti-CD44 and loaded with Im (GNP-HCIm) were synthesized. Lung fibroblasts (LFs) and alveolar macrophages from bronchoalveolar lavage fluids of SSc-ILD patients were cultured in presence of nanoparticles. GNP-HCIm significantly inhibited proliferation and viability inducing apoptosis of LFs and effectively reduced IL-8 release, viability and M2 polarization in alveolar macrophages. Anti-fibrotic effect of tracheal instilled GNP-HCIm was evaluated on bleomycin lung fibrosis mouse model comparing effect with common route of Im administration. GNP-HCIm were able to reduce significantly lung fibrotic changes and collagen deposition. Finally, electron microscopy revealed the presence of GNPs inside alveolar macrophages. These data support the use of GNPs locally administered in the development of new therapeutic approaches to SSc-ILD.

Bronchiolitis Obliterans Syndrome is the major determinant of the graft function loss after lung transplantation, but its pathogenesis is still incompletely understood and currently available therapeutic strategies are poorly effective. A deeper understanding of its pathogenic mechanisms is crucial for the development of new strategies to prevent and treat this devastating complication. In this study, we focused on the mesenchymal stromal cells, recently recognized as BOS key effectors, and our primary aim was to identify their epigenetic determinants, such as histone modifications and non-coding RNA regulation, which could contribute to their differentiation in myofibroblasts. Interestingly, we identified a deregulated expression of histone deacetylases and methyltransferases, and a microRNA-epigenetic regulatory network, which could represent novel targets for anti-fibrotic therapy. We validated our results in vitro , in a cell model of fibrogenesis, confirming the epigenetic involvement in this process and paving the way for a new application for epigenetic drugs.

Bronchiolitis obliterans syndrome (BOS), caused by lung allograft-derived mesenchymal cells’ abnormal proliferation and extracellular matrix deposition, is the main cause of lung allograft rejection. In this study, a mild one-step ionotropic gelation method was set up to nanoencapsulate the everolimus, a key molecule in allograft organ rejection prevention, into hyaluronic acid-decorated chitosan-based nanoparticles. Rationale was the selective delivery of everolimus into lung allograft-derived mesenchymal cells; these cells are characterized by the CD44-overexpressing feature, and hyaluronic acid has proven to be a natural selective CD44-targeting moiety. The optimal process conditions were established by a design of experiment approach (full factorial design) aiming at the control of the nanoparticle size (≤200 nm), minimizing the size polydispersity (PDI 0.171 ± 0.04), and at the negative ζ potential maximization (−30.9 mV). The everolimus was successfully loaded into hyaluronic acid-decorated chitosan-based nanoparticles (95.94 ± 13.68 μg/100 mg nanoparticles) and in vitro released in 24 h. The hyaluronic acid decoration on the nanoparticles provided targetability to CD44-overexpressing mesenchymal cells isolated from bronchoalveolar lavage of BOS-affected patients. The mesenchymal cells’ growth tests along with the nanoparticles uptake studies, at 37 °C and 4 °C, respectively, demonstrated a clear improvement of everolimus inhibitory activity when it is encapsulated in hyaluronic acid-decorated chitosan-based nanoparticles, ascribable to their active uptake mechanism.

Objectives. Diabetic neuropathy is the most common complication of diabetes. The idea of alterations in energy metabolism in diabetes is emerging. The biogenic antioxidant R(+)-thioctic acid has been successfully used in the treatment of diabetic polyneuropathic (DPN) patients. Methods. The effects of R(+)-thioctic acid (1 tablet, 1.6 g) administration were evaluated in 12 DPN patients at baseline and at 15, 30, 60, and 120 administration days throughout the assessment of oxidative stress (OxS); ROS production rate by electron paramagnetic resonance (EPR) technique; and oxidative damage biomarkers (thiobarbituric acid reactive substances (TBARS) and protein carbonyls (PC)), electroneurography (ENG) and visual analogue scale. Results. Supplementation induced significant changes (p<0.05) at 30 and 60 days. ROS production rate up to −16%; TBARS (−31%), PC (−38%), and TAC up to +48%. Motor nerve conduction velocity in SPE and ulnar nerves (+22% and +16%) and sensor conduction velocity in sural and median nerves (+22% and +5%). Patients reported a general wellness sensation improvement (+35%) at 30 days: lower limb pain sensation (−40%) and upper limbs (−23%). Conclusion. The results strongly indicate that an increased antioxidant capacity plays an important role in OxS, nerve conduction velocity, pain, and general wellness improvement. Nevertheless, the effects of the antioxidant compound were found positive up to 60 days. Then, a hormesis effect was observed. Novelty of the research would be a challenge for investigators to carefully address issues, including dose range factors, appropriate administration time, and targeting population to counteract possible “boomerang effects.” The great number of monitored parameters would firmly stress these conclusions.

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

Malignant Pleural Mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis and continuously increasing incidence due to widespread exposure to asbestos. Novel approaches for MPM management could take advantage of the intrapleural administration of drugs. Our group has recently developed a novel nanoplatform using gold nanoparticles (GNPs) aimed at the local treatment of lung diseases [Cova E et al. 2015]. We already proved that engineered GNPs loaded with pemetrexed and specifically decorated with the anti-CD146 expressed by MPM cells, were highly effective in inhibiting cancer cells [Stella GM et al. Eur Respir J 2015 46: OA5003]. The MAP kinase pathway is known to regulate proliferation and survival of tumor cells, including MPM [Myioshy S et al. 2012]. Clinical improvement of selumetinib slowed down due to reported adverse events [Jänne PA et al, 2013]. Here, we aimed at investigating the therapeutic efficacy of selumetinib on MPM. Therefore, we preliminary tested selumetinib on two different MPM cell lines, MSTO-211H and H2452, representative of biphasic and epithelioid subtypes respectively (Fig.1). We found that selumetinib was efficacy in inhibiting MSTO-211H line as evidenced by half maximal inhibitory concentration (IC50) of 1.59 uM after 96 h incubation. However, selumetinib was almost ineffective in inhibiting H2452 cells by using the same drug concentrations suggesting a resistance to MEK inhibitors, as already observed for melanoma cell lines (Emery et al., 2009). At least in selected cases, selumetinib might be an useful drug to be loaded by functionalized targeted GNPs

Our work has the objective to develop and provide a new therapeutic approach to rare respiratory disease with poor prognosis. These diseases are united by the fact of being rare diseases, defined orphan, with a very poor prognosis and limited treatment options. Furthermore, they share the possibility to apply a local treatment with the advantage of decreased unwanted biodistribution and systemic toxicity. Bronchiolitis obliterans (BO) is a disease characterized by fibrotic obliteration of the small airways that occurs in response to inflammatory and immunological insults. Malignant pleural mesothelioma (MPM) is a rare malignant tumor that originates from the pleura, strongly associated with environmental exposure to asbestos. Our approach consists in the creation of nanocarriers (gold nanoparticles, GNPs), which can be loaded with specific anti-proliferative drugs, specifically targeted to the cells responsible of the two pathological processes (fibroblastoid-like mesenchymal cells in BO, malignant mesothelioma cells in MPM) and suitable for administration by local street (inhaled or intrapleural). First, we isolated, cultured and phenotyped primary cells from patients from BO and MPM. Once you have identified some targets (CD44 for the BO and CD146 for MPM) we designed a nanotool loaded with the specific drugs (everolimus or pemetrexed) and decorated with monoclonal antibody on the surface. We performed experiments in vitro on primary cultures of pathological cells and we demonstrated that these nanoparticles were able to penetrate specifically in target cells expressing the specific receptor and not in other types of normal cells tested, except for the alveolar macrophage which presented the tendency to absorb the nanoparticles, also the not functionalized ones. The anti-proliferative, pro-apoptotic and anti-inflammatory action of these nanoparticles was tested in vitro. We have also conducted experiments in animals to prove the lack of both pulmonary and extrapulmonary toxicity following administration of nanoparticles by inhalation.

The role of CD4+CD25highCD127− T-reg cells in solid-organ Transplant (Tx) acceptance has been extensively studied. In previous studies on kidney and liver recipients, peripheral T-reg cell counts were associated to graft survival, while in lung Tx, there is limited evidence for similar findings. This study aims to analyze long term peripheral kinetics of T-reg-cells in a cohort of lung recipients and tests its association to several clinical variables.

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.

We demonstrated that targeted-gold-nanoparticles loaded with everolimus (GNP-HCe) were effective in vitro inhibiting mesenchymal cells isolated from bronchoalveolar lavage of BOS patients. Here, we investigated the effect of GNPs on inflammatory cells, given that a stimulating effect is strongly unwanted. Macrophages were obtained from BAL by adhesion procedure, lymphocytes and neutrophils from peripheral blood by Lympholyte. Cells were incubated with GNP-HC (targeted GNP without everolimus), GNP-HCe, everolimus (EV) and medium. IL-8 was assayed by ELISA, IFN-g, IL-17 and IL-10 by Elispot. Elastase release was evaluated by enzymatic test. Apoptosis, CD3+CD4+, CD3+CD8+ populations and CCR7 expression were analyzed by flow cytometry, cell viability by MTT test, and mitochondria membrane potential by JC-1 fluorescence. Reactive oxygen species (ROS) production was assayed by EPR spectroscopy. GNP-HCe did not stimulate IL-8 secretion by macrophages but reduced cell viability (p<0,05). IFN-g, IL-17 and IL-10 produced by lymphocytes decreased after GNP-HCe incubation (p<0,01) while apoptosis was stimulated (p<0,01). GNP-HCe reduced the CD3+CD4+ and CD3+CD8+ population (p<0.01). CCR7 expression on CD4+ and CD8+ cells was increased (p<0,01). Elastase release was unchanged by GNP-HCe incubation while apoptotic rate increased (p<0,05). Membrane potential and ROS production of both macrophages and neutrophils was significantly affected by GNP-HCe (p<0,001 and p<0,05, respectively). Our study confirm that GNP-HCe are able to exert its therapeutic action without activating immune effectors with positive implications on the translational potential of this therapeutic strategy.

A complex computational pipeline to perform enrichment analysis of miRNAs in pathways was applied to the study of post lung transplant Bronchiolitis Obliterans Syndrome (BOS). The analysis considers the full set of miRNAs annotated in miRBase (version 21), and applies different filtering approaches and statistical analyses to reduce this set and to score the candidate miRNAs with potential involvement in BOS development (Figure 1). In order to validate results of the computational analysis we analyzed, by ISH, two highly ranked miRNAs, miR-21 and miR-34a, in a set of lung tissue samples obtained from 3 patients with BOS and in mesenchymal cells from BAL of lung recipients in stable conditions, or with BOS (0p-3) and, as control, normal skin fibroblasts and primary mesenchymal cells obtained from patients with COP. Preliminary ISH results demonstrated a dysregulation of these miRNAs and its localization in BOS lesions in human transplanted lungs. miR-21 showed a net overexpression in BOS lesions (Figure 2): it was primarily expressed in fibroblasts and in activated epithelial cells in all human BOS cases, while it was absent in normal lungs, thus clearly demonstrating upregulation in BOS; miR-34a was clearly expressed in BOS proliferating fibroblasts and in several non-inflammatory cell types, normal bronchiolar and alveolar epithelia and reactive pneumocytes. miR-21 expression in primary mesenchymal cell samples correlated with ISH results. The results we obtained open the possibility of identifying relevant key molecules involved in BOS which will be used as additional biomarkers and possible therapeutic targets and of gaining insight into the complex pathogenic network of this disease.