Publications by authors named "Stefano Gatti"

90 Publications

Atelectasis, Shunt, and Worsening Oxygenation Following Reduction of Respiratory Rate in Healthy Pigs Undergoing ECMO: An Experimental Lung Imaging Study.

Front Physiol 2021 9;12:663313. Epub 2021 Apr 9.

Department of Anesthesia, Critical Care and Emergency, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Reducing the respiratory rate during extracorporeal membrane oxygenation (ECMO) decreases the mechanical power, but it might induce alveolar de-recruitment. Dissecting de-recruitment due to lung edema vs. the fraction due to hypoventilation may be challenging in injured lungs. We characterized changes in lung physiology (primary endpoint: development of atelectasis) associated with progressive reduction of the respiratory rate in healthy animals on ECMO. Six female pigs underwent general anesthesia and volume control ventilation (Baseline: PEEP 5 cmHO, Vt 10 ml/kg, I:E = 1:2, FiO 0.5, rate 24 bpm). Veno-venous ECMO was started and respiratory rate was progressively reduced to 18, 12, and 6 breaths per minute (6-h steps), while all other settings remained unchanged. ECMO blood flow was kept constant while gas flow was increased to maintain stable PaCO. At Baseline (without ECMO) and toward the end of each step, data from quantitative CT scan, electrical impedance tomography, and gas exchange were collected. Increasing ECMO gas flow while lowering the respiratory rate was associated with an increase in the fraction of non-aerated tissue (i.e., atelectasis) and with a decrease of tidal ventilation reaching the gravitationally dependent lung regions ( = 0.009 and = 0.018). Intrapulmonary shunt increased ( < 0.001) and arterial PaO decreased ( < 0.001) at lower rates. The fraction of non-aerated lung was correlated with longer expiratory time spent at zero flow ( = 0.555, = 0.011). Progressive decrease of respiratory rate coupled with increasing CO removal in mechanically ventilated healthy pigs is associated with development of lung atelectasis, higher shunt, and poorer oxygenation.
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http://dx.doi.org/10.3389/fphys.2021.663313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063114PMC
April 2021

NDP-MSH treatment recovers marginal lungs during ex vivo lung perfusion (EVLP).

Peptides 2021 Apr 15;141:170552. Epub 2021 Apr 15.

Center for Preclinical Research, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20100, Milan, Italy.

The increasing use of marginal lungs for transplantation encourages novel approaches to improve graft quality. Melanocortins and their receptors (MCRs) exert multiple beneficial effects in pulmonary inflammation. We tested the idea that treatment with the synthetic α-melanocyte-stimulating hormone analogue [Nle4,D-Phe7]-α-MSH (NDP-MSH) during ex vivo lung perfusion (EVLP) could exert positive influences in lungs exposed to different injuries. Rats were assigned to one of the following protocols (N = 10 each): 1) ischemia/reperfusion (IR) or 2) cardiac death (CD) followed by ex vivo perfusion. NDP-MSH treatment was performed in five rats of each protocol before lung procurement and during EVLP. Pulmonary function and perfusate concentration of gases, electrolytes, metabolites, nitric-oxide, mediators, and cells were assessed throughout EVLP. ATP content and specific MCR expression were investigated in perfused lungs and in biopsies collected from rats in resting conditions (Native, N = 5). NDP-MSH reduced the release of inflammatory mediators in perfusates of both the IR and the CD groups. Treatment was likewise associated with a lesser amount of leukocytes (IR: p = 0.034; CD: p = 0.002) and reduced lactate production (IR: p = 0.010; CD: p = 0.008). In lungs exposed to IR injury, the NDP-MSH group showed increased ATP content (p = 0.040) compared to controls. In CD lungs, a significant improvement of vascular (p = 0.002) and airway (Ppeak: p < 0.001, compliance: p < 0.050, pO2: p < 0.001) parameters was observed. Finally, the expression of MC1R and MC5R was detected in both native and ex vivo-perfused lungs. The results indicate that NDP-MSH administration preserves lung function through broad positive effects on multiple pathways and suggest that exploitation of the melanocortin system during EVLP could improve reconditioning of marginal lungs before transplantation.
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http://dx.doi.org/10.1016/j.peptides.2021.170552DOI Listing
April 2021

Defective dystrophic thymus determines degenerative changes in skeletal muscle.

Nat Commun 2021 04 8;12(1):2099. Epub 2021 Apr 8.

Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Unit of Neurology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Milan, Italy.

In Duchenne muscular dystrophy (DMD), sarcolemma fragility and myofiber necrosis produce cellular debris that attract inflammatory cells. Macrophages and T-lymphocytes infiltrate muscles in response to damage-associated molecular pattern signalling and the release of TNF-α, TGF-β and interleukins prevent skeletal muscle improvement from the inflammation. This immunological scenario was extended by the discovery of a specific response to muscle antigens and a role for regulatory T cells (Tregs) in muscle regeneration. Normally, autoimmunity is avoided by autoreactive T-lymphocyte deletion within thymus, while in the periphery Tregs monitor effector T-cells escaping from central regulatory control. Here, we report impairment of thymus architecture of mdx mice together with decreased expression of ghrelin, autophagy dysfunction and AIRE down-regulation. Transplantation of dystrophic thymus in recipient nude mice determine the up-regulation of inflammatory/fibrotic markers, marked metabolic breakdown that leads to muscle atrophy and loss of force. These results indicate that involution of dystrophic thymus exacerbates muscular dystrophy by altering central immune tolerance.
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http://dx.doi.org/10.1038/s41467-021-22305-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032677PMC
April 2021

Modified pediatric Lung Ultrasound Score compared with computed tomography for assessment of lung aeration in children.

Minerva Anestesiol 2021 Feb 17. Epub 2021 Feb 17.

Department of Emergency and Intensive Care, San Gerardo Hospital, Monza, Italy.

Background: Lung ultrasound can be used to assess lung density and aeration at the bedside. A few authors have investigated scores based on the ultrasonographic interstitial syndrome for this purpose, but none have compared them with the gold standard computed tomography in children.

Methods: Children < 10 kilograms undergoing a chest computed tomography for clinical purposes at a tertiary hospital Pediatric Intensive Care Unit were enrolled in the study. An ultrasound scan was performed shortly after computed tomography. Each hemithorax was divided in 6 zones, and each zone was scored: 1 = no B lines; 2 = < 3 B lines; 3 > 3 well separated B lines; 4 = crowded, coalescent B lines; 5 = white lung; 6 = consolidation. The pediatric Lung Ultrasound Score was obtained by adding all zones. Interobserver variation for two separate operators was calculated.

Results: Ten children, median age 95 days (range 23 - 721), were enrolled. Mean pediatric Lung Ultrasound Score had a significant correlation with lung density (ρ = 0.68) and percentage of hypoaerated lung (ρ = 0.51). Median density and percentage of hypoaerated lung increased along the ultrasound patterns values (p < 0.05) although not all patterns were significantly different from adjacent ones in the pairwise comparison. Interobserver variability in scoring of ultrasonographic patterns was moderate.

Conclusions: The pediatric Lung Ultrasound Score correlates with lung density and percentage of hypoaerated lung measured with computed tomography.
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http://dx.doi.org/10.23736/S0375-9393.21.15155-7DOI Listing
February 2021

Activation of Melanocortin Receptors as a Potential Strategy to Reduce Local and Systemic Reactions Induced by Respiratory Viruses.

Front Endocrinol (Lausanne) 2020 10;11:569241. Epub 2020 Dec 10.

Center for Preclinical Research, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

The clinical hallmarks of infections caused by critical respiratory viruses consist of pneumonia, which can progress to acute lung injury (ALI), and systemic manifestations including hypercoagulopathy, vascular dysfunction, and endotheliitis. The disease outcome largely depends on the immune response produced by the host. The bio-molecular mechanisms underlying certain dire consequences of the infection partly arise from an aberrant production of inflammatory molecules, an event denoted as "cytokine storm". Therefore, in addition to antiviral therapies, molecules able to prevent the injury caused by cytokine excess are under investigation. In this perspective, taking advantage of melanocortin peptides and their receptors, components of an endogenous modulatory system that exerts marked anti-inflammatory and immunomodulatory influences, could be an effective therapeutic strategy to control disease evolution. Exploiting the melanocortin system using natural or synthetic ligands can form a realistic basis to counteract certain deleterious effects of respiratory virus infections. The central and peripheral protective actions exerted following melanocortin receptor activation could allow dampening the harmful events that trigger the cytokine storm and endothelial dysfunction while sustaining the beneficial signals required to elicit repair mechanisms. The long standing evidence for melanocortin safety encourages this approach.
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http://dx.doi.org/10.3389/fendo.2020.569241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758465PMC
January 2021

Increased ratio of P[v-a]CO to C[a-v]O without global hypoxia: the case of metformin-induced lactic acidosis.

Respir Physiol Neurobiol 2021 03 14;285:103586. Epub 2020 Nov 14.

Department of Anaesthesia and Intensive Care Units, Humanitas Clinical and Research Center - IRCCS, Rozzano Milan, Italy; Department of Biomedical Sciences, Humanitas University, Pieve Emanuele Milan, Italy. Electronic address:

The ratio of venoarterial CO tension to arteriovenous O content difference (P[v-a]CO/C[a-v]O) increases when lactic acidosis is due to inadequate oxygen supply (hypoxia); we aimed to verify whether it also increases when lactic acidosis develops because of mitochondrial dysfunction (dysoxia) with constant oxygen delivery. Twelve anaesthetised, mechanically ventilated pigs were intoxicated with IV metformin (4.0 to 6.4 g over 2.5 to 4.0 h). Saline and norepinephrine were used to preserve oxygen delivery. Lactate and P[v-a]CO/C[a-v]O were measured every one or two hours (arterial and mixed venous blood). During metformin intoxication, lactate increased from 0.8 (0.6-0.9) to 8.5 (5.0-10.9) mmol/l (p < 0.001), even if oxygen delivery remained constant (from 352 ± 78 to 343 ± 97 ml/min, p = 0.098). P[v-a]CO/C[a-v]O increased from 1.6 (1.2-1.8) to 2.3 (1.9-3.2) mmHg/ml/dl (p = 0.004). The intraclass correlation coefficient between lactate and P[v-a]CO/C[a-v]O was 0.72 (p < 0.001). We conclude that P[v-a]CO/C[a-v]O increases when lactic acidosis is due to dysoxia. Therefore, a high P[v-a]CO/C[a-v]O may not discriminate hypoxia from dysoxia as the cause of lactic acidosis.
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http://dx.doi.org/10.1016/j.resp.2020.103586DOI Listing
March 2021

The RNA-dependent DNA methylation pathway is required to restrict expression to specify a single female germ cell precursor in .

Development 2020 12 13;147(23). Epub 2020 Dec 13.

Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy

In higher plants, the female germline is formed from the megaspore mother cell (MMC), a single cell in the premeiotic ovule. Previously, it was reported that mutants in the RNA-dependent DNA methylation (RdDM) pathway might be involved in restricting the female germline to a single nucellus cell. We show that the DRM methyltransferase double mutant also presents ectopic enlarged cells, consistent with supernumerary MMC-like cells. In wild-type ovules, MMC differentiation requires SPOROCYTELESS/NOZZLE (SPL/NZZ), as demonstrated by the mutant failing to develop an MMC. We address the poorly understood upstream regulation of SPL/NZZ in ovules, showing that the RdDM pathway is important to restrict SPL/NZZ expression. In , and mutants, SPL/NZZ is expressed ectopically, suggesting that the multiple MMC-like cells observed might be attributable to the ectopic expression of SPL/NZZ. We show that the ovule identity gene, , directly regulates and expression in the ovule and therefore indirectly regulates SPL/NZZ expression. A model is presented describing the network required to restrict SPL/NZZ expression to specify a single MMC.
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http://dx.doi.org/10.1242/dev.194274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758631PMC
December 2020

PTX3 Predicts Myocardial Damage and Fibrosis in Duchenne Muscular Dystrophy.

Front Physiol 2020 19;11:403. Epub 2020 May 19.

Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Milan, Italy.

Pentraxin 3 (PTX3) is a main component of the innate immune system by inducing complement pathway activation, acting as an inflammatory mediator, coordinating the functions of macrophages/dendritic cells and promoting apoptosis/necrosis. Additionally, it has been found in fibrotic regions co-localizing with collagen. In this work, we wanted to investigate the predictive role of PTX3 in myocardial damage and fibrosis of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive disease caused by mutations of the dystrophin gene that affects muscular functions and strength and accompanying dilated cardiomyopathy. Here, we expound the correlation of PTX3 cardiac expression with age and Toll-like receptors (TLRs)/interleukin-1 receptor (IL-1R)-MyD88 inflammatory markers and its modulation by the so-called alarmins IL-33, high-mobility group box 1 (HMGB1), and S100β. These findings suggest that cardiac levels of PTX3 might have prognostic value and potential in guiding therapy for DMD cardiomyopathy.
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http://dx.doi.org/10.3389/fphys.2020.00403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248204PMC
May 2020

Mboat7 down-regulation by hyper-insulinemia induces fat accumulation in hepatocytes.

EBioMedicine 2020 Feb 12;52:102658. Epub 2020 Feb 12.

Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Ospedale Policlinico via F Sforza 35, 20122 Milano, Italy; Translational Medicine, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Italy. Electronic address:

Background: Naturally occurring variation in Membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), encoding for an enzyme involved in phosphatidylinositol acyl-chain remodelling, has been associated with fatty liver and hepatic disorders. Here, we examined the relationship between hepatic Mboat7 down-regulation and fat accumulation.

Methods: Hepatic MBOAT7 expression was surveyed in 119 obese individuals and in experimental models. MBOAT7 was acutely silenced by antisense oligonucleotides in C57Bl/6 mice, and by CRISPR/Cas9 in HepG2 hepatocytes.

Findings: In obese individuals, hepatic MBOAT7 mRNA decreased from normal liver to steatohepatitis, independently of diabetes, inflammation and MBOAT7 genotype. Hepatic MBOAT7 levels were reduced in murine models of fatty liver, and by hyper-insulinemia. In wild-type mice, Mboat7 was down-regulated by refeeding and insulin, concomitantly with insulin signalling activation. Acute hepatic Mboat7 silencing promoted hepatic steatosis in vivo and enhanced expression of fatty acid transporter Fatp1. MBOAT7 deletion in hepatocytes reduced the incorporation of arachidonic acid into phosphatidylinositol, consistently with decreased enzymatic activity, determining the accumulation of saturated triglycerides, enhanced lipogenesis and FATP1 expression, while FATP1 deletion rescued the phenotype.

Interpretation: MBOAT7 down-regulation by hyper-insulinemia contributes to hepatic fat accumulation, impairing phosphatidylinositol remodelling and up-regulating FATP1.

Funding: LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute RF-2016-02,364,358, Ricerca corrente Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico; LV and AG received funding from the European Union Programme Horizon 2020 (No. 777,377) for the project LITMUS-"Liver Investigation: Testing Marker Utility in Steatohepatitis". MM was supported by Fondazione Italiana per lo Studio del Fegato (AISF) 'Mario Coppo' fellowship.
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http://dx.doi.org/10.1016/j.ebiom.2020.102658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026742PMC
February 2020

Procurement and ex-situ perfusion of isolated slaughterhouse-derived livers as a model of donors after circulatory death.

ALTEX 2019 12 12. Epub 2019 Dec 12.

Center for Preclinical Research, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy.

Ex-situ machine perfusion (MP) techniques are increasingly used in clinical settings, especially on grafts derived from donors after cardiac death (DCD). However, comprehension of biological effects elicited during MP are largely unknown and a substantial number of animal studies are presently focused on this topic. The aim of the present study was to describe a model of DCD based on ex-situ perfusion of liver grafts derived from animals dedicated to food production. Procurement took place within a slaughterhouse facility. A clinically fashioned closed circuit normothermic MP (NMP) was built up. Autologous blood-enriched perfusion fluid was adopted. Perfusate and tissue samples were collected to asses NMP functionality. Grafts were classified as transplantable (LT-G) or not (n-LT) according to clinical criteria, while histopathological analysis was used to confirm graft viability. After cold storage, the liver grafts were connected to the NMP. During the rewarming phase, temperature and flows were progressively increased to reach target values. At the end of NMP, 4 grafts were classified as LT-G and 3 nLT-G. Histology confirmed absence of major damage in LT-G, while diffuse necrosis appeared in nLT-G. Interestingly, in nLT-G an early impairment of hepatocyte respiratory chain, leading to cell necrosis and graft non-viability, was documented for the first time. These parameters, together with indocyanine-green dye and citrate clearance could contribute to graft evaluation in clinical settings. In conclusion, this model provides a promising and reproducible method to replace dedicated experimental animals in DCD and MP research, in line with the 3Rs principles.
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http://dx.doi.org/10.14573/altex.1909131DOI Listing
December 2019

Human Red Blood Cells as Oxygen Carriers to Improve Ex-Situ Liver Perfusion in a Rat Model.

J Clin Med 2019 Nov 8;8(11). Epub 2019 Nov 8.

Center for Preclinical Research, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 20019 Milan, Italy.

Ex-situ machine perfusion (MP) has been increasingly used to enhance liver quality in different settings. Small animal models can help to implement this procedure. As most normothermic MP (NMP) models employ sub-physiological levels of oxygen delivery (DO), the aim of this study was to investigate the effectiveness and safety of different DO, using human red blood cells (RBCs) as oxygen carriers on metabolic recovery in a rat model of NMP. Four experimental groups (n = 5 each) consisted of (1) native (untreated/control), (2) liver static cold storage (SCS) 30 min without NMP, (3) SCS followed by 120 min of NMP with Dulbecco-Modified-Eagle-Medium as perfusate (DMEM), and (4) similar to group 3, but perfusion fluid was added with human RBCs (hematocrit 15%) (BLOOD). Compared to DMEM, the BLOOD group showed increased liver DO ( = 0.008) and oxygen consumption ( V O ˙ ) ( < 0.001); lactate clearance ( < 0.001), potassium ( < 0.001), and glucose ( = 0.029) uptake were enhanced. ATP levels were likewise higher in BLOOD relative to DMEM ( = 0.031). V O ˙ and DO were highly correlated ( < 0.001). Consistently, the main metabolic parameters were directly correlated with DO and V O ˙ . No human RBC related damage was detected. In conclusion, an optimized DO significantly reduces hypoxic damage-related effects occurring during NMP. Human RBCs can be safely used as oxygen carriers.
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http://dx.doi.org/10.3390/jcm8111918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912657PMC
November 2019

mir-101-3p Downregulation Promotes Fibrogenesis by Facilitating Hepatic Stellate Cell Transdifferentiation During Insulin Resistance.

Nutrients 2019 Oct 29;11(11). Epub 2019 Oct 29.

General Medicine and Metabolic Diseases, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano 20122, Italy.

Insulin resistance (IR) and microRNAs (miRNAs), which regulate cell-to-cell communication between hepatocytes and hepatic stellate cells (HSCs), may intertwine in nonalcoholic fatty liver disease (NAFLD) pathogenesis. The aim of this study was to evaluate whether epigenetics and environmental factors interact to promote progressive NAFLD during IR. We examined the miRNA signature in insulin receptor haploinsufficient (InsR+/-) and wild-type (wt) HSCs by RNAseq ( = 4 per group). Then, we evaluated their impact in an IR-NASH (nonalcoholic steatohepatitis) model (InsR+/- mice fed standard or methionine choline deficient (MCD) diet, = 10 per group) and in vitro. InsR+/- HSCs displayed 36 differentially expressed miRNAs ( < 0.05 vs. wt), whose expression was then analyzed in the liver of InsR+/- mice fed an MCD diet. We found that miR-101-3p negatively associated with both InsR+/- genotype and MCD ( < 0.05) and the histological spectrum of liver damage ( < 0.01). miR-101-3p was reduced in InsR+/- hepatocytes and HSCs and even more in InsR+/- cells exposed to insulin (0.33 µM) and fatty acids (0.25 mM), resembling the IR-NASH model. Conversely, insulin induced miR-101-3p expression in wt cells but not in InsR+/- ones ( < 0.05). In conclusion, IR combined with diet-induced liver injury favors miR-101-3p downregulation, which may promote progressive NAFLD through HSC and hepatocyte transdifferentiation and proliferation.
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http://dx.doi.org/10.3390/nu11112597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893471PMC
October 2019

Extracorporeal Chloride Removal by Electrodialysis. A Novel Approach to Correct Acidemia.

Am J Respir Crit Care Med 2020 04;201(7):799-813

Department of Medical Physiopathology and Transplants, University of Milan, Milan, Italy.

Acidemia is a severe condition among critically ill patients. Despite lack of evidence, sodium bicarbonate is frequently used to correct pH; however, its administration is burdened by several side effects. We hypothesized that the reduction of plasma chloride concentration could be an alternative strategy to correct acidemia. To evaluate feasibility, safety, and effectiveness of a novel strategy to correct acidemia through extracorporeal chloride removal by electrodialysis. Ten swine (six treated and four control animals) were sedated, mechanically ventilated and connected to an extracorporeal electrodialysis device capable of selectively removing chloride. In random order, an arterial pH of 7.15 was induced either through reduction of ventilation (respiratory acidosis) or through lactic acid infusion (metabolic acidosis). Acidosis was subsequently sustained for 12-14 hours. In treatment pigs, soon after reaching target acidemia, electrodialysis was started to restore pH. During respiratory acidosis, electrodialysis reduced plasma chloride concentration by 26 ± 5 mEq/L within 6 hours (final pH = 7.36 ± 0.04). Control animals exhibited incomplete and slower compensatory response to respiratory acidosis (final pH = 7.29 ± 0.03;  < 0.001). During metabolic acidosis, electrodialysis reduced plasma chloride concentration by 15 ± 3 mEq/L within 4 hours (final pH = 7.34 ± 0.07). No effective compensatory response occurred in control animals (final pH = 7.11 ± 0.08;  < 0.001). No complications occurred. We described the first application of an extracorporeal system targeted to correct severe acidemia by lowering plasma chloride concentration. Extracorporeal chloride removal by electrodialysis proved to be feasible, safe, and effective. Further studies are warranted to assess its performance in the presence of impaired respiratory and renal functions.
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http://dx.doi.org/10.1164/rccm.201903-0538OCDOI Listing
April 2020

Mesenchymal stem cell-derived extracellular vesicles improve the molecular phenotype of isolated rat lungs during ischemia/reperfusion injury.

J Heart Lung Transplant 2019 12 24;38(12):1306-1316. Epub 2019 Aug 24.

Center for Preclinical Research, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milan, Italy.

Background: Lung ischemia/reperfusion (IR) injury contributes to the development of severe complications in patients undergoing transplantation. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) exert beneficial actions comparable to those of MSCs without the risks of the cell-based strategy. This research investigated EV effects during IR injury in isolated rat lungs.

Methods: An established model of 180-minutes ex vivo lung perfusion (EVLP) was used. At 60 minutes EVs (n = 5) or saline (n = 5) were administered. Parallel experiments used labeled EVs to determine EV biodistribution (n = 4). Perfusate samples were collected to perform gas analysis and to assess the concentration of nitric oxide (NO), hyaluronan (HA), inflammatory mediators, and leukocytes. Lung biopsies were taken at 180 minutes to evaluate HA, adenosine triphosphate (ATP), gene expression, and histology.

Results: Compared with untreated lungs, EV-treated organs showed decreased vascular resistance and a rise of perfusate NO metabolites. EVs prevented the reduction in pulmonary ATP caused by IR. Increased medium-high-molecular-weight HA was detected in the perfusate and in the lung tissue of the IR + EV group. Significant differences in cell count on perfusate and tissue samples, together with induction of transcription and synthesis of chemokines, suggested EV-dependent modulation of leukocyte recruitment. EVs upregulated genes involved in the resolution of inflammation and oxidative stress. Biodistribution analysis showed that EVs were retained in the lung tissue and internalized within pulmonary cells.

Conclusions: This study shows multiple novel EV influences on pulmonary energetics, tissue integrity, and gene expression during IR. The use of cell-free therapies during EVLP could constitute a valuable strategy for reconditioning and repair of injured lungs before transplantation.
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http://dx.doi.org/10.1016/j.healun.2019.08.016DOI Listing
December 2019

Graft Portal Vein Thrombosis Before Liver Transplant.

Transplantation 2020 01;104(1):e44-e45

General and Liver Transplant Surgery Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico Milan, Milan, Italy.

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http://dx.doi.org/10.1097/TP.0000000000002919DOI Listing
January 2020

CSF transplantation of a specific iPSC-derived neural stem cell subpopulation ameliorates the disease phenotype in a mouse model of spinal muscular atrophy with respiratory distress type 1.

Exp Neurol 2019 11 21;321:113041. Epub 2019 Aug 21.

Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), University of Milan, Italy. Electronic address:

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a genetic motor neuron disease affecting infants. This condition is caused by mutations in the IGHMBP2 gene and currently has no cure. Stem cell transplantation is a potential therapeutic strategy for motor neuron diseases such as SMARD1, exerting beneficial effects both by replacing cells and by providing support to endogenous motor neurons. In this work, we demonstrate that human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) selected for the expression of specific markers, namely, Lewis X, CXCR4 and beta 1 integrin, and pretreated with neurotrophic factors and apoptosis/necroptosis inhibitors were able to effectively migrate and engraft into the host parenchyma after administration into the cerebrospinal fluid in a SMARD1 mouse model. We were able to detect donor cells in the ventral horn of the spinal cord and observe improvements in neuropathological features, particularly preservation of the integrity of the motor unit, that were correlated with amelioration of the SMARD1 disease phenotype in terms of neuromuscular function and lifespan. This minimally invasive stem cell approach can confer major advantages in the context of cell-mediated therapy for patients with neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.expneurol.2019.113041DOI Listing
November 2019

Driving Pressure Is Associated with Outcome during Assisted Ventilation in Acute Respiratory Distress Syndrome.

Anesthesiology 2019 09;131(3):594-604

From the Department of Medicine and Surgery, University of Milan-Bicocca, Monza, Italy (G.B., A.G., S.S., S.G., G.F.) Department of Emergency and Intensive Care, San Gerardo Hospital, Monza, Italy (G.B., A.G., S.S., S.G., G.F.) Departments of Critical Care Medicine and Anesthesia, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada (B.P.K.) Department of Anesthesia, Critical Care and Emergency Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy (A.P.).

What We Already Know About This Topic: Higher driving pressure during controlled mechanical ventilation is known to be associated with increased mortality in patients with acute respiratory distress syndrome.Whereas patients with acute respiratory distress syndrome are initially managed with controlled mechanical ventilation, as they improve, they are transitioned to assisted ventilation. Whether higher driving pressure assessed during pressure support (assisted) ventilation can be reliably assessed and whether higher driving pressure is associated with worse outcomes in patients with acute respiratory distress syndrome has not been well studied.

What This Article Tells Us That Is New: This study shows that in the majority of adult patients with acute respiratory distress syndrome, both driving pressure and respiratory system compliance can be reliably measured during pressure support (assisted) ventilation.Higher driving pressure measured during pressure support (assisted) ventilation significantly associates with increased intensive care unit mortality, whereas peak inspiratory pressure does not.Lower respiratory system compliance also significantly associates with increased intensive care unit mortality.

Background: Driving pressure, the difference between plateau pressure and positive end-expiratory pressure (PEEP), is closely associated with increased mortality in patients with acute respiratory distress syndrome (ARDS). Although this relationship has been demonstrated during controlled mechanical ventilation, plateau pressure is often not measured during spontaneous breathing because of concerns about validity. The objective of the present study is to verify whether driving pressure and respiratory system compliance are independently associated with increased mortality during assisted ventilation (i.e., pressure support ventilation).

Methods: This is a retrospective cohort study conducted on 154 patients with ARDS in whom plateau pressure during the first three days of assisted ventilation was available. Associations between driving pressure, respiratory system compliance, and survival were assessed by univariable and multivariable analysis. In patients who underwent a computed tomography scan (n = 23) during the stage of assisted ventilation, the quantity of aerated lung was compared with respiratory system compliance measured on the same date.

Results: In contrast to controlled mechanical ventilation, plateau pressure during assisted ventilation was higher than the sum of PEEP and pressure support (peak pressure). Driving pressure was higher (11 [9-14] vs. 10 [8-11] cm H2O; P = 0.004); compliance was lower (40 [30-50] vs. 51 [42-61] ml · cm H2O; P < 0.001); and peak pressure was similar, in nonsurvivors versus survivors. Lower respiratory system compliance (odds ratio, 0.92 [0.88-0.96]) and higher driving pressure (odds ratio, 1.34 [1.12-1.61]) were each independently associated with increased risk of death. Respiratory system compliance was correlated with the aerated lung volume (n = 23, r = 0.69, P < 0.0001).

Conclusions: In patients with ARDS, plateau pressure, driving pressure, and respiratory system compliance can be measured during assisted ventilation, and both higher driving pressure and lower compliance are associated with increased mortality.
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http://dx.doi.org/10.1097/ALN.0000000000002846DOI Listing
September 2019

Central role of the β-cell in driving regression of diabetes after liver transplantation in cirrhotic patients.

J Hepatol 2019 05 21;70(5):954-962. Epub 2019 Jan 21.

Diabetes Service, Endocrinology and Metabolic Diseases Unit, IRCCS "Cà Granda - Ospedale Maggiore Policlinico" Foundation, and Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy. Electronic address:

Background & Aims: Diabetes occurring as a direct consequence of loss of liver function is usually characterized by non-diabetic fasting plasma glucose (FPG) and haemoglobin A (HbA) levels and should regress after orthotopic liver transplantation (OLT). This observational, longitudinal study investigated the relationship between the time-courses of changes in all 3 direct determinants of glucose regulation, i.e., β-cell function, insulin clearance and insulin sensitivity, and diabetes regression after OLT.

Methods: Eighty cirrhotic patients with non-diabetic FPG and HbA levels underwent an extended oral glucose tolerance test (OGTT) before and 3, 6, 12 and 24 months after OLT. The OGTT data were analysed with a mathematical model to estimate derivative control (DC) and proportional control (PC) of β-cell function and insulin clearance (which determine insulin bioavailability), and with the Oral Glucose Insulin Sensitivity (OGIS)-2 h index to estimate insulin sensitivity.

Results: At baseline, 36 patients were diabetic (45%) and 44 were non-diabetic (55%). Over the 2-year follow-up, 23 diabetic patients (63.9%) regressed to non-diabetic glucose regulation, whereas 13 did not (36.1%); moreover, 4 non-diabetic individuals progressed to diabetes (9.1%), whereas 40 did not (90.9%). Both DC and PC increased in regressors (from month 3 and 24, respectively) and decreased in progressors, whereas they remained stable in non-regressors and only PC decreased in non-progressors. Insulin clearance increased in all groups, apart from progressors. Likewise, OGIS-2 h improved at month 3 in all groups, but thereafter it continued to improve only in regressors, whereas it returned to baseline values in the other groups.

Conclusions: Increased insulin bioavailability driven by improved β-cell function plays a central role in favouring diabetes regression after OLT, in the presence of a sustained improvement of insulin sensitivity.

Lay Summary: Diabetes occurring in cirrhosis as a direct consequence of loss of liver function should regress after transplantation of a new functioning liver, though the pathophysiological mechanisms are unclear. This is the first study evaluating the contribution of all 3 direct determinants of insulin-dependent glucose regulation using a sophisticated mathematical model. Results show that β-cell function is the key process governing favourable or detrimental changes in glucose regulation in cirrhotic patients undergoing transplantation, pointing to the need to develop therapies to sustain β-cell function in these individuals.

Trial Registration: ClinicalTrials.gov, NCT02038517.
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http://dx.doi.org/10.1016/j.jhep.2019.01.015DOI Listing
May 2019

Proteome Investigation of Rat Lungs subjected to Ex Vivo Perfusion (EVLP).

Molecules 2018 11 22;23(12). Epub 2018 Nov 22.

Department of Pathophysiology and Transplantation, Università degli Studi di Milano, and Internal Medicine Department, Respiratory Unit and Adult Cystic Fibrosis Center, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy.

Ex vivo lung perfusion (EVLP) is an emerging procedure that allows organ preservation, assessment and reconditioning, increasing the number of marginal donor lungs for transplantation. However, physiological and airflow measurements are unable to unveil the molecular mechanisms responsible of EVLP beneficial effects on lung graft and monitor the proper course of the treatment. Thus, it is urgent to find specific biomarkers that possess these requirements but also accurate and reliable techniques that identify them. The purpose of this study is to give an overview on the potentiality of shotgun proteomic platforms in characterizing the status and the evolution of metabolic pathways during EVLP in order to find new potential EVLP-related biomarkers. A nanoLC-MS/MS system was applied to the proteome analysis of lung tissues from an optimized rat model in three experimental groups: native, pre- and post-EVLP. Technical and biological repeatability were evaluated and, together with clustering analysis, underlined the good quality of data produced. In-house software and bioinformatics tools allowed the label-free extraction of differentially expressed proteins among the three examined conditions and the network visualization of the pathways mainly involved. These promising findings encourage further proteomic investigations of the molecular mechanisms behind EVLP procedure.
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http://dx.doi.org/10.3390/molecules23123061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321151PMC
November 2018

ABO blood types and major outcomes in patients with acute hypoxaemic respiratory failure: A multicenter retrospective cohort study.

PLoS One 2018 25;13(10):e0206403. Epub 2018 Oct 25.

School of Medicine and Surgery, University of Milan-Bicocca, Monza, Italy.

Introduction: ABO blood type A was reported to correlate with an increased risk of acute respiratory distress syndrome (ARDS) in white patients with severe sepsis and major trauma compared with patients with other blood types. Information regarding ABO phenotypes and major outcomes in patients with ARDS is unavailable. The primary aim was to determine the relationship between ABO blood type A and intensive care unit (ICU) mortality in patients with acute hypoxemic respiratory failure (AHRF). The secondary aim was to describe the association between ABO blood type A and ICU length of stay (LOS) in this study population.

Methods: In a multicenter, retrospective cohort study, we collected the clinical records of patients admitted from January 2012 to December 2014 in five ICUs of Northern Italy. We included adult white patients admitted to the ICU who were diagnosed with AHRF requiring mechanical ventilation.

Results: The electronic records of 1732 patients with AHRF were reviewed. The proportion of patients with ABO blood type A versus other blood types was 39.9% versus 60.1%. ICU mortality (25%) and ICU LOS (median [interquartile range], 5 [2-12] days) were not different when stratified by ABO blood type (ICU mortality, overall p value = 0.905; ICU LOS, overall p value = 0.609). SAPSII was a positive predictor of ICU mortality (odds ration [OR], 32.80; 95% confidence interval [CI], 18.80-57.24; p < 0.001) and ICU LOS (β coefficient, 0.55; 95% CI, 0.35-0.75; p < 0.001) at multivariate analyses, whereas ABO blood type did not predict ICU outcome when forced into the model.

Conclusion: ABO blood type did not correlate with ICU mortality and ICU LOS in adult patients with AHRF who were mechanically ventilated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206403PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201964PMC
April 2019

Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

FASEB J 2018 10 2;32(10):5532-5549. Epub 2018 May 2.

Center for Preclinical Investigation, Dipartimento di Anestesia, Rianimazione ed Emergenza Urgenza, Fondazione Istituto Di Ricovero e Cura a Carattere Scientifico Ca' Granda - Ospedale Maggiore Policlinico, Milan, Italy.

Despite increasing clinical adoption, biologic influences of ex vivo lung perfusion (EVLP) remain insufficiently elucidated. The aim of the current study was to investigate biomolecular changes induced by EVLP in rat lungs. EVLP was maintained for 180 min. Hyaluronan, mediators, and cells were assessed in the perfusate. Gene expression, signaling pathways, and ATP content were investigated in lung tissue. EVLP induced the release of medium-high molecular weight hyaluronan and transcription of hyaluronan synthases ( P < 0.001). Increasing concentrations of inflammatory mediators were detected in the perfusate ( P < 0.001). Perfused lungs exhibited a distinctive transcriptional signature compared with organs examined before or after surgery/procurement ( P = 0.003). Up-regulated genes were involved in inflammation and its regulation, apoptosis/survival, heat shock, and oxidative stress response ( q = 0). Down-regulated genes were related to lymphocyte function ( q = 0). The NF-κB, signal transducer and activator of transcription 3, ERK1/2, p38, Akt, and stress-activated protein kinase/JNK signaling pathways were modulated by EVLP ( P < 0.05). Most of these biomolecular changes were examined and confirmed in additional experiments that were performed in lungs procured from donation after cardiocirculatory death after 180 min of warm ischemia. The current study demonstrates that EVLP broadly affects the lung biomolecular phenotype. These findings improve our comprehension of the effects exerted by the procedure and encourage additional research in preclinical models to implement therapeutic interventions.-Lonati, C., Bassani, G. A., Brambilla, D., Leonardi, P., Carlin, A., Faversani, A., Gatti, S., Valenza, F. Influence of ex vivo perfusion on the biomolecular profile of rat lungs.
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http://dx.doi.org/10.1096/fj.201701255RDOI Listing
October 2018

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

J Allergy Clin Immunol 2018 11 31;142(5):1537-1547.e8. Epub 2018 Jan 31.

INGM-National Institute of Molecular Genetics "Romeo ed Enrica Invernizzi" Milan, Milan, Italy. Electronic address:

Background: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T1) cells but is also produced by CD25 regulatory T (Treg) cells.

Objective: We aimed to identify and characterize human intestinal T1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs).

Methods: CD4 T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed.

Results: Intestinal T1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5PD-1 T1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ T1 cells, but not IL-7 receptor-positive T cells or CD25 Treg cells, showed lower IL-10 expression in patients with IBDs. T1 cells were responsive to IL-23, and IFN-γ T1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression.

Conclusions: We provide the first ex vivo characterization of human intestinal T1 cells. Selective downregulation of IL-10 by IFN-γ T1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.
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http://dx.doi.org/10.1016/j.jaci.2017.12.984DOI Listing
November 2018

Bedside selection of positive end-expiratory pressure by electrical impedance tomography in hypoxemic patients: a feasibility study.

Ann Intensive Care 2017 Dec 20;7(1):76. Epub 2017 Jul 20.

Department of Emergency and Intensive Care, San Gerardo Hospital, Via Pergolesi 33, Monza, Italy.

Background: Positive end-expiratory pressure (PEEP) is a key element of mechanical ventilation. It should optimize recruitment, without causing excessive overdistension, but controversy exists on the best method to set it. The purpose of the study was to test the feasibility of setting PEEP with electrical impedance tomography in order to prevent lung de-recruitment following a recruitment maneuver. We enrolled 16 patients undergoing mechanical ventilation with PaO/FiO <300 mmHg. In all patients, under constant tidal volume (6-8 ml/kg) PEEP was set based on the PEEP/FiO table proposed by the ARDS network (PEEP). We performed a recruitment maneuver and monitored the end-expiratory lung impedance (EELI) over 10 min. If the EELI signal decreased during this period, the recruitment maneuver was repeated and PEEP increased by 2 cmHO. This procedure was repeated until the EELI maintained a stability over time (PEEP).

Results: The procedure was feasible in 87% patients. PEEP was higher than PEEP (13 ± 3 vs. 9 ± 2 cmHO, p < 0.001). PaO/FiO improved during PEEP and driving pressure decreased. Recruited volume correlated with the decrease in driving pressure but not with oxygenation improvement. Finally, regional alveolar hyperdistention and collapse was reduced in dependent lung layers and increased in non-dependent lung layers.

Conclusions: In hypoxemic patients, a PEEP selection strategy aimed at stabilizing alveolar recruitment guided by EIT at the bedside was feasible and safe. This strategy led, in comparison with the ARDSnet table, to higher PEEP, improved oxygenation and reduced driving pressure, allowing to estimate the relative weight of overdistension and recruitment.
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http://dx.doi.org/10.1186/s13613-017-0299-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519511PMC
December 2017

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.

Clin Sci (Lond) 2017 06 7;131(12):1301-1315. Epub 2017 Jun 7.

Internal Medicine, Fondazione IRCCS Ca' Granda Ospedale Policlinico Milano, Milan, Italy

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.
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http://dx.doi.org/10.1042/CS20170175DOI Listing
June 2017

Ex Vivo Lung Perfusion in the Rat: Detailed Procedure and Videos.

PLoS One 2016 9;11(12):e0167898. Epub 2016 Dec 9.

Center for Surgical Research, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy.

Ex vivo lung perfusion (EVLP) is a promising procedure for evaluation, reconditioning, and treatment of marginal lungs before transplantation. Small animal models can contribute to improve clinical development of this technique and represent a substantial platform for bio-molecular investigations. However, to accomplish this purpose, EVLP models must sustain a prolonged reperfusion without pharmacological interventions. Currently available protocols only partly satisfy this need. The aim of the present research was accomplishment and optimization of a reproducible model for a protracted rat EVLP in the absence of anti-inflammatory treatment. A 180 min, uninjured and untreated perfusion was achieved through a stepwise implementation of the protocol. Flow rate, temperature, and tidal volume were gradually increased during the initial reperfusion phase to reduce hemodynamic and oxidative stress. Low flow rate combined with open atrium and protective ventilation strategy were applied to prevent lung damage. The videos enclosed show management of the most critical technical steps. The stability and reproducibility of the present procedure were confirmed by lung function evaluation and edema assessment. The meticulous description of the protocol provided in this paper can enable other laboratories to reproduce it effortlessly, supporting research in the EVLP field.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167898PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148015PMC
July 2017

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

Mol Ther 2016 Nov 10;24(11):1898-1912. Epub 2016 Aug 10.

Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Unit of Neurology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Milan, Italy. Electronic address:

Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.
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http://dx.doi.org/10.1038/mt.2016.162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154478PMC
November 2016

A New Transgenic Mouse Model of Heart Failure and Cardiac Cachexia Raised by Sustained Activation of Met Tyrosine Kinase in the Heart.

Biomed Res Int 2016 19;2016:9549036. Epub 2016 May 19.

Department of Oncology, University of Turin, 10126 Turin, Italy.

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States, and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the preclinical level.
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http://dx.doi.org/10.1155/2016/9549036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889800PMC
February 2017

Role of Strain Rate in the Pathogenesis of Ventilator-Induced Lung Edema.

Crit Care Med 2016 09;44(9):e838-45

1Dipartimento di Anestesia, Rianimazione ed Emergenza Urgenza, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy.2Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi, Milan, Italy.3Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy.4Centro di Ricerche Chirurgiche Precliniche, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Università degli Studi, Milan, Italy.5U.O.C. Anatomia Patologica, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy.6Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy.7Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi, Milan, Italy.

Objective: Lungs behave as viscoelastic polymers. Harms of mechanical ventilation could then depend on not only amplitude (strain) but also velocity (strain rate) of lung deformation. Herein, we tested this hypothesis.

Design: Laboratory investigation.

Setting: Animal unit.

Subjects: Thirty healthy piglets.

Interventions: Two groups of animals were ventilated for 54 hours with matched lung strains (ratio between tidal volume and functional residual capacity) but different lung strain rates (ratio between strain and inspiratory time). Individual strains ranged between 0.6 and 3.5 in both groups. Piglets ventilated with low strain rates had an inspiratory-to-expiratory time ratio of 1:2-1:3. Those ventilated with high strain rates had much lower inspiratory-to-expiratory time ratios (down to 1:9). Respiratory rate was always 15 breaths/min. Lung viscoelastic behavior, with ventilator setting required per protocol, was "quantified" as dynamic respiratory system hysteresis (pressure-volume loop [in Joules]) and stress relaxation (airway pressure drop during an end-inspiratory pause [in cm H2O]). Primary outcome was the occurrence of pulmonary edema within 54 hours.

Measurements And Main Results: On average, the two study groups were ventilated with well-matched strains (2.1 ± 0.9 vs 2.1 ± 0.9; p = 0.864) but different strain rates (1.8 ± 0.8 vs 4.6 ± 1.5 s; p < 0.001), dynamic respiratory system hysteresis (0.6 ± 0.3 vs 1.4 ± 0.8 J; p = 0.001), and stress relaxation (3.1 ± 0.9 vs 5.0 ± 2.3 cm H2O; p = 0.008). The prevalence of pulmonary edema was 20% among piglets ventilated with low strain rates and 73% among those ventilated with high strain rates (p = 0.010).

Conclusions: High strain rate is a risk factor for ventilator-induced pulmonary edema, possibly because it amplifies lung viscoelastic behavior.
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http://dx.doi.org/10.1097/CCM.0000000000001718DOI Listing
September 2016

Cardiac concentric hypertrophy promoted by activated Met receptor is mitigated in vivo by inhibition of Erk1,2 signalling with Pimasertib.

J Mol Cell Cardiol 2016 04 26;93:84-97. Epub 2016 Feb 26.

Department of Oncology, University of Turin, 10126 Turin, Italy. Electronic address:

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.
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http://dx.doi.org/10.1016/j.yjmcc.2016.02.017DOI Listing
April 2016

Mechanical Power and Development of Ventilator-induced Lung Injury.

Anesthesiology 2016 May;124(5):1100-8

From the Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy (M.C., M.G., C.C., D.M., I.A., M.A., A.C., M.B., C.M., K.N., M.G., G.L.V., P.P., P.C.); Centro di Ricerche Precliniche, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy (D.D., S.G.); Department of Anesthesiology and Intensive Care Medicine, Georg-August-University Goettingen, Goettingen, Germany (L.G.); and Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy (V.V., N.G.).

Background: The ventilator works mechanically on the lung parenchyma. The authors set out to obtain the proof of concept that ventilator-induced lung injury (VILI) depends on the mechanical power applied to the lung.

Methods: Mechanical power was defined as the function of transpulmonary pressure, tidal volume (TV), and respiratory rate. Three piglets were ventilated with a mechanical power known to be lethal (TV, 38 ml/kg; plateau pressure, 27 cm H2O; and respiratory rate, 15 breaths/min). Other groups (three piglets each) were ventilated with the same TV per kilogram and transpulmonary pressure but at the respiratory rates of 12, 9, 6, and 3 breaths/min. The authors identified a mechanical power threshold for VILI and did nine additional experiments at the respiratory rate of 35 breaths/min and mechanical power below (TV 11 ml/kg) and above (TV 22 ml/kg) the threshold.

Results: In the 15 experiments to detect the threshold for VILI, up to a mechanical power of approximately 12 J/min (respiratory rate, 9 breaths/min), the computed tomography scans showed mostly isolated densities, whereas at the mechanical power above approximately 12 J/min, all piglets developed whole-lung edema. In the nine confirmatory experiments, the five piglets ventilated above the power threshold developed VILI, but the four piglets ventilated below did not. By grouping all 24 piglets, the authors found a significant relationship between the mechanical power applied to the lung and the increase in lung weight (r = 0.41, P = 0.001) and lung elastance (r = 0.33, P < 0.01) and decrease in PaO2/FIO2 (r = 0.40, P < 0.001) at the end of the study.

Conclusion: In piglets, VILI develops if a mechanical power threshold is exceeded.
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http://dx.doi.org/10.1097/ALN.0000000000001056DOI Listing
May 2016