Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

Mass Transport via In-Plane Nanopores in Graphene Oxide Membranes.

Angstrom-confined solvents in 2D laminates can travel through interlayer spacings, through gaps between adjacent sheets, and via in-plane pores. Among these, experimental access to investigate the mass transport through in-plane pores is lacking. Our experiments allow an understanding of this mass transport via the controlled variation of oxygen functionalities, size and density of in-plane pores in graphene oxide membranes. Contrary to expectations, our transport experiments show that higher in-plane pore densities may not necessarily lead to higher water permeability. We observed that membranes with a high in-plane pore density but a low amount of oxygen functionalities exhibit a complete blockage of water. However, when water-ethanol mixtures with a weaker hydrogen network are used, these membranes show an enhanced permeation. Our combined experimental and computational results suggest that the transport mechanism is governed by the attraction of the solvents toward the pores with functional groups and hindered by the strong hydrogen network of water formed under angstrom confinement.

Dalbavancin Boosts the Ability of Neutrophils to Fight Methicillin-Resistant Staphylococcus aureus.

Polymorphonuclear leukocytes (PMNs) are the most important cell type involved in the early nonspecific host response to bacterial pathogens. Staphylococcus aureus has evolved mechanisms to evade immune responses that contribute to its persistence in PMNs, and acquired resistance to several antimicrobials. Additionally, methicillin-resistant S. aureus (MRSA) is one of the most common causes of acute bacterial skin and skin-structure infections (ABSSSIs). Dalbavancin (DBV), a lipoglycopeptide, is indicated for the treatment of ABSSSIs, and has a broad spectrum of action against most microorganisms. Here, we sought to determine the effect of DBV on the neutrophil killing of MRSA and its potential immunomodulating activity. Our results revealed that DBV boosts MRSA killing by acting on both bacteria and PMNs. DBV pre-treatment of PMNs did not change the respiratory burst or degranulation, while an increased trend in neutrophil extracellular traps-associated elastase and in the production of TNFα and CXCL8 was revealed. In parallel, DBV caused a delay in the apoptosis of MRSA-infected neutrophils. In conclusion, we demonstrated a cooperative effect between the antimicrobial properties of PMNs and DBV, thus owing to their immunomodulatory activity. In the choice of the treatment management of serious S. aureus infections, DBV should be considered as an outstanding option since it reinforces PMNs pathogen clearance capability by exerting its effect directly, not only on MRSA but also on neutrophils.

Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cell Activation and Infectibility That Amplifies the Angiogenic Process.

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.

Atomistic Simulations in Surface Chemistry to Interpret Scanning Probe Microscopy Images: A Short Review.

Atomistic simulations are a powerful tool to explain and guide experimental investigations, but there are cases where a clear correspondence is difficult to obtain. While the theoretical framework to get the static picture of the equilibrium structures in vacuum is well-established, it is challenging to correctly model them in operando conditions (at the right experimental temperature, pH and pressure). In this short review the main theoretical approaches are briefly presented, supported by selected case studies where the structural and dynamical properties of different systems are investigated. A successful match with the experimental data is accomplished by choosing the proper level of theory in order to describe the structure under study in the most accurate and realistic way.

Platelets, diabetes and myocardial ischemia/reperfusion injury.

Mechanisms underlying the pathogenesis of ischemia/reperfusion injury are particularly complex, multifactorial and highly interconnected. A complex and entangled interaction is also emerging between platelet function, antiplatelet drugs, coronary diseases and ischemia/reperfusion injury, especially in diabetic conditions. Here we briefly summarize features of antiplatelet therapy in type 2 diabetes (T2DM). We also treat the influence of T2DM on ischemia/reperfusion injury and how anti-platelet therapies affect post-ischemic myocardial damage through pleiotropic properties not related to their anti-aggregating effects. miRNA-based signature associated with T2DM and its cardiovascular disease complications are also briefly considered. Influence of anti-platelet therapies and different effects of healthy and diabetic platelets on ischemia/reperfusion injury need to be further clarified in order to enhance patient benefits from antiplatelet therapy and revascularization. Here we provide insight on the difficulty to reduce the cardiovascular risk in diabetic patients and report novel information on the cardioprotective role of widely used anti-aggregant drugs.

Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM-1 expression.

DCs are powerful antigen-presenting cells central in the orchestration of innate and acquired immunity. DC development, migration, and activities are intrinsically linked to the microenvironment. DCs migrate through pathologic tissues before reaching their final destination in the lymph nodes. Hypoxia, a condition of low partial oxygen pressure, is a common feature of many pathologic situations, capable of modifying DC phenotype and functional behavior. We studied human monocyte-derived immature DCs generated under chronic hypoxic conditions (H-iDCs). We demonstrate by gene expression profiling the upregulation of a cluster of genes coding for antigen-presentation, immunoregulatory, and pattern recognition receptors, suggesting a stimulatory role for hypoxia on iDC immunoregulatory functions. In particular, we show that H-iDCs express triggering receptor expressed on myeloid cells(TREM-1), a member of the Ig superfamily of immunoreceptors and an amplifier of inflammation. This effect is reversible because H-iDC reoxygenation results in TREM-1 down-modulation. TREM-1 engagement promotes upregulation of T-cell costimulatory molecules and homing chemokine receptors, typical of mature DCs, and increases the production of proinflammatory, Th1/Th17-priming cytokines/chemokines, resulting in increased T-cell responses. These results suggest that TREM-1 induction by the hypoxic microenvironment represents a mechanism of regulation of Th1-cell trafficking and activation by iDCs differentiated at pathologic sites.

Atomic structure of a spinel-like transition Al2O3(100) surface.

We study a crystalline epitaxial alumina thin film with the characteristics of a spinel-type transition Al2O3(100) surface by using atom-resolved noncontact atomic force microscopy and density functional theory. It is shown that the films are terminated by an Al-O layer rich in Al vacancies, exhibiting a strong preference for surface hydroxyl group formation in two configurations. The transition alumina films are crystalline and perfectly stable in ambient atmospheres, a quality which is expected to open the door to new fundamental studies of the surfaces of transition aluminas.

IFN-ß expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-ß. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-ß in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-ß mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-ß transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-ß promoter, revealed that IFN-ß mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-ß mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.

Antimicrobial Efficacy of a Vegetable Oil Plasticizer in PVC Matrices.

The growing prevalence of bacterial and viral infections, highlighted by the recent COVID-19 pandemic, urgently calls for new antimicrobial strategies. To this end, we have synthesized and characterized a novel fatty acid epoxy-ester plasticizer for polymers, named GDE. GDE is not only sustainable and user-friendly but also demonstrates superior plasticizing properties, while its epoxy components improve the heat stability of PVC-based matrices. A key feature of GDE is its ability to confer antimicrobial properties to surfaces. Indeed, upon contact, this material can effectively kill enveloped viruses, such as herpes simplex virus type 1 (HSV-1) and the ß-coronavirus prototype HCoV-OC43, but it is ineffective against nonenveloped viruses like human adenovirus (HAdV). Further analysis using transmission electron microscopy (TEM) on HSV-1 virions exposed to GDE showed significant structural damage, indicating that GDE can interfere with the viral envelope, potentially causing leakage. Moreover, GDE demonstrates antibacterial activity, albeit to a lesser extent, against notorious pathogens such as Staphylococcus aureus and Escherichia coli. Overall, this newly developed plasticizer shows significant potential as an antimicrobial agent suitable for use in both community and healthcare settings to curb the spread of infections caused by microorganisms contaminating physical surfaces.

Understanding water transport through graphene-based nanochannels via experimental control of slip length.

The water transport along graphene-based nanochannels has gained significant interest. However, experimental access to the influence of defects and impurities on transport poses a critical knowledge gap. Here, we investigate the water transport of cation intercalated graphene oxide membranes. The cations act as water-attracting impurities on the channel walls. Via water transport experiments, we show that the slip length of the nanochannels decay exponentially with the hydrated diameter of the intercalated cations, confirming that water transport is governed by the interaction between water molecules and the impurities on the channel wall. The exponential decay of slip length approximates non-slip conditions. This offers experimental support for the use of the Hagen-Poiseuille equation in graphene-based nanochannels, which was previously only confirmed by simulations. Our study gives valuable feedback to theoretical predictions of the water transport along graphene-based channels with water-attracting impurities.

Norepinephrine and Serotonin Can Modulate the Behavior of the Probiotic Enterococcus faecium NCIMB10415 towards the Host: Is a Putative Surface Sensor Involved?

The human gut microbiota has co-evolved with humans by exchanging bidirectional signals. This study aims at deepening the knowledge of this crucial relationship by analyzing phenotypic and interactive responses of the probiotic Enterococcus faecium NCIMB10415 (E. faecium SF68) to the top-down signals norepinephrine (NE) and serotonin (5HT), two neuroactive molecules abundant in the gut. We treated E. faecium NCIMB10415 with 100 µM NE and 50 µM 5HT and tested its ability to form static biofilm (Confocal Laser Scanning Microscopy), adhere to the Caco-2/TC7 monolayer, affect the epithelial barrier function (Transepithelial Electrical Resistance) and human dendritic cells (DC) maturation, differentiation, and cytokines production. Finally, we evaluated the presence of a putative hormone sensor through in silico (whole genome sequence and protein modelling) and in vitro (Micro-Scale Thermophoresis) analyses. The hormone treatments increase biofilm formation and adhesion on Caco-2/TC7, as well as the epithelial barrier function. No differences concerning DC differentiation and maturation between stimulated and control bacteria were detected, while an enhanced TNF-α production was observed in NE-treated bacteria. Investigations on the sensor support the hypothesis that a two-component system on the bacterial surface can sense 5HT and NE. Overall, the data demonstrate that E. faecium NCIMB10415 can sense both NE and 5HT and respond accordingly.

Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14.

Activin A is a dimeric protein, member of the transforming growth factor (TGF)-beta family that plays a crucial role in wound repair and in fetal tolerance. Emerging evidence also proposes activin A as a key mediator in inflammation. This study reports that activin A induces the directional migration of immature myeloid dendritic cells (iDCs) through the activation of ALK4 and ActRIIA receptor chains. Conversely, activin A was not active on plasmacytoid dendritic cells (DCs) or mature myeloid DCs. iDC migration to activin A was phosphatidylinositol 3-kinase gamma-dependent, Bordetella pertussis toxin- and cycloheximide-sensitive, and was inhibited by M3, a viral-encoded chemokine-binding protein. In a real-time video microscopy-based migration assay, activin A induced polarization of iDCs, but not migration. These characteristics clearly differentiated the chemotactic activities of activin A from TGF-beta and classic chemokines. By the use of combined pharmacologic and low-density microarray analysis, it was possible to define that activin-A-induced migration depends on the selective and polarized release of 2 chemokines, namely CXC chemokine ligands 12 and 14. This study extends the proinflammatory role of activin A to DC recruitment and provides a cautionary message about the reliability of the in vitro chemotaxis assays in discriminating direct versus indirect chemotactic agonists.

Survival and migration of human dendritic cells are regulated by an IFN-alpha-inducible Axl/Gas6 pathway.

Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In this study, we show that Axl expression is induced by IFN-alpha during human dendritic cell (DC) differentiation from monocytes (IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-alpha. This effect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axl down-regulation on the surface of human IFN-alpha-treated DC correlates with an increased proteolytic cleavage of Axl and with elevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axl expression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced chemotaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidence that Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. This encourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunity characterized by high levels of IFN-alpha.

Enhanced Antimicrobial and Antibiofilm Effect of New Colistin-Loaded Human Albumin Nanoparticles.

Multidrug-resistant (MDR) Gram-negative bacteria (GNB), such as Acinetobacter and Klebsiella, are responsible for severe hospital-acquired infections. Colistin, despite its toxicity and low tissue penetration, is considered the last resort antibiotic against these microorganisms. Of concern, the use of Colistin has recently been compromised by the emergence of Colistin resistance. Herein, we developed a new formulation consisting of multifunctional chitosan-coated human albumin nanoparticles for the delivery of Colistin (Col/haNPs). Col/haNPs were in vitro characterized for encapsulation efficiency, drug release, stability and cytotoxicity and were evaluated for antibacterial activity against MDR GNB (Acinetobacter baumannii and Klebsiella pneumoniae). Col/haNPs showed sizes lower than 200 nm, high encapsulation efficiency (98.65%) and prolonged in vitro release of Colistin. The safety of the nanoformulation was demonstrated by a negligible cytotoxicity on human fibroblasts and hemolytic activity. Col/haNPs evidenced a high antibacterial effect with a significant decrease in MIC values compared to free Colistin, in particular against Col-resistant strains with a pronounced decline of bacterial growth over time. Moreover, Col/haNPs exhibited an inhibitory effect on biofilm formation that was 4 and 60 fold higher compared to free Colistin, respectively for Colistin susceptible and resistant A. baumannii. Our findings suggest that Col/haNPs could represent a promising Colistin nanocarrier with high antimicrobial activity on MDR GNB.

Bisphenol A and S in the Urine of Newborns: Plastic for Non-Food Use Still without Rules.

The aim of the present study was to assess the effects of bisphenol (BP) exposure on pregnancy and neonatal life. We have (a) determined BP (BPA and BPS) concentration levels in a group of newborns and their mothers; (b) identified factors, habits, and devices possibly responsible for BP uptake; and (c) determined the effect of BP exposure. No significant correlations were detected between maternal and neonatal BP concentration levels. In newborns, positive correlations between pacifier use and BPS total (p = 0.04) and free BPS (p = 0.03) concentrations were detected. A significant correlation was also found between oral glucose administration and concentration levels of free BPA (p < 0.05). Our study points to a central role of lifestyle, hospital procedures, and neonatal devices in inducing BP exposure, especially during the perinatal period. This is the first report of BP contamination in newborns due to widely non-alimentary products designed for newborn care, such as glucose-solution containers for BPA and pacifiers for BPS. Further studies are advocated in order to clarify both the impact of other BP forms on human health and development, as well as potential BPA exposure sources during neonatal and childhood life.

Selective Proton Transport for Hydrogen Production Using Graphene Oxide Membranes.

Graphene oxide has shown exceptional properties in terms of water permeability and filtration characteristics. Here the suitability of graphene oxide membranes for the spatial separation of hydronium and hydroxide ions after photocatalytic water splitting is demonstrated. Instead of relying on classical size exclusion by adjusting the membrane laminates' interlayer spacings, nonmodified graphene oxide is used to exploit the presence of its natural functional groups and surface charges for filtration. Despite a significantly larger interlayer spacing inside the membrane compared with the size of the hydrated radii of the ions, highly asymmetric transport behavior and a 6 times higher mobility for hydronium than for hydroxide are observed. DFT simulations reveal that hydroxide ions are more prone to interact and stick to the functional groups of graphene oxide, while diffusion of hydronium ions through the membrane is less impeded and aligns well with the concept of the Grotthuss mechanism.

Inhibition of Human Neutrophil Functions In Vitro by Multiple Sclerosis Disease-Modifying Therapies.

There is a growing optimism about the potential of new disease-modifying therapies (DMTs) in the management of relapsing-remitting multiple sclerosis (RRMS) patients. However, this initial enthusiasm has been tempered by evidence indicating that multiple sclerosis (MS) patients undergoing DMT may be at higher risk of developing infections through incompletely understood mechanisms. As neutrophils provide the first line of defense against pathogens, here we have compared the effects of some of the commonly used MS DMTs (i.e., moderate-efficacy injective, first-line: interferonß-1b (IFNß-1b), glatiramer acetate (GA); and high-efficacy, second-line: fingolimod (FTY) and natalizumab (NAT)) on the in vitro viability and functions of neutrophils isolated from healthy subjects. All the DMTs tested impaired the ability of neutrophils to kill Klebsiella pneumoniae, whereas none of them affected the rate of neutrophil apoptosis or CD11b and CD62L cell surface expression. Intriguingly, only FTY exposure negatively affected K. pneumoniae-induced production of reactive oxygen species (ROS) in polymorphonuclear leukocytes (PMNs). Furthermore, neutrophils exposed to K. pneumoniae secreted enhanced amounts of CXCL8, IL-1ß and TNF-α, which were differentially regulated following DMT pretreatment. Altogether, these findings suggest that DMTs may increase the susceptibility of MS patients to microbial infections, in part, through inhibition of neutrophil functions. In light of these data, we recommend that the design of personalized therapies for RRMS patients should take into account not just the mechanism of action of the chosen DMT but also the potential risk of infection associated with the administration of such therapeutic compounds to this highly vulnerable population.

CSF1R Is Required for Differentiation and Migration of Langerhans Cells and Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by tissue accumulation of CD1a+CD207+ LCH cells. In LCH, somatic mutations of the BRAF V600E gene have been detected in tissue LCH cells, bone marrow CD34+ hematopoietic stem cells, circulating CD14+ monocytes, and BDCA1+ myeloid dendritic cells (DC). Targeting BRAF V600E in clonal Langerhans cells (LC) and their precursors is a potential treatment option for patients whose tumors have the mutation. The development of mouse macrophages and LCs is regulated by the CSF1 receptor (CSF1R). In patients with diffuse-type tenosynovial giant cell tumors, CSF1R inhibition depletes tumor-associated macrophages (TAM) with therapeutic efficacy; however, CSF1R signaling in LCs and LCH has not been investigated. We found through IHC and flow cytometry that CSF1R is normally expressed on human CD1a+CD207+ LCs in the epidermis and stratified epithelia. LCs that were differentiated from CD14+ monocytes, BDCA1+ DCs, and CD34+ cord blood progenitors expressed CSF1R that was downregulated upon maturation. Immature LCs migrated toward CSF1, but not IL34. Administration of the c-FMS/CSF1R kinase inhibitors GW2580 and BLZ945 significantly reduced human LC migration. In LCH clinical samples, LCH cells (including BRAF V600E cells) and TAMs retained high expression of CSF1R. We also detected the presence of transcripts for its ligand, CSF1, but not IL34, in all tested LCH cases. CSF1R and CSF1 expression in LCH, and their role in LC migration and differentiation, suggests CSF1R signaling blockade as a candidate rational approach for treatment of LCH, including the BRAF V600E and wild-type forms of the disease.

Impact of imipenem concentration in lung perfusate and tissue biopsy during clinical ex-vivo lung perfusion of high-risk lung donors.

BACKGROUND: Normothermic ex-vivo lung perfusion (EVLP) limits organ donor shortage by potentially using high-risk donor lungs. Microbial burden reduction has been demonstrated after EVLP using antibiotic prophylaxis with imipenem. However, no data have been published on the clinical consequences of the potential residual bacterial burden. METHODS: Imipenem concentration was measured every hour (T0 to T6) in the lung perfusate and at the end of EVLP (Tf) in biopsies. The antimicrobial activity of perfusate at T1 and Tf against E. coli and K. pneumoniae was evaluated. Lungs were distinguished: no bacterial species in recipients and donors (donor-/recipient-); bacterial species isolated from donors and not from recipients (donor+/recipient-); same bacterial species in both recipients and donors (donor+/recipient+). Interleukin 6 (IL-6) and IL-8 concentrations in lung perfusate, clinical pulmonary infection score (CPIS) and primary graft dysfunction (PGD) were evaluated. RESULTS: Imipenem concentration in perfusate decreased over time. T1 and Tf perfusates exhibited bactericidal activity against E. coli and K. pneumoniae. Overall, T1 perfusates yielded higher bactericidal titers (BTs) than Tf. The donor+/recipient+ group (26% of cases) had higher IL-6 and IL-8 in perfusate and higher CPIS. CONCLUSIONS: Recipients with the same bacterial species isolated in their donors had higher risk of pulmonary inflammation and early post-transplant pneumonia. Improvements in antimicrobial strategies during EVLP are warranted to minimize the consequences of donor associated respiratory infection.