Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Urinary phenotyping of SARS-CoV-2 infection connects clinical diagnostics with metabolomics and uncovers impaired NAD+ pathway and SIRT1 activation.

OBJECTIVES: The stratification of individuals suffering from acute and post-acute SARS-CoV-2 infection remains a critical challenge. Notably, biomarkers able to specifically monitor viral progression, providing details about patient clinical status, are still not available. Herein, quantitative metabolomics is progressively recognized as a useful tool to describe the consequences of virus-host interactions considering also clinical metadata. METHODS: The present study characterized the urinary metabolic profile of 243 infected individuals by quantitative nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LC-MS). Results were compared with a historical cohort of noninfected subjects. Moreover, we assessed the concentration of recently identified antiviral nucleosides and their association with other metabolites and clinical data. RESULTS: Urinary metabolomics can stratify patients into classes of disease severity, with a discrimination ability comparable to that of clinical biomarkers. Kynurenines showed the highest fold change in clinically-deteriorated patients and higher-risk subjects. Unique metabolite clusters were also generated based on age, sex, and body mass index (BMI). Changes in the concentration of antiviral nucleosides were associated with either other metabolites or clinical variables. Increased kynurenines and reduced trigonelline excretion indicated a disrupted nicotinamide adenine nucleotide (NAD+) and sirtuin 1 (SIRT1) pathway. CONCLUSIONS: Our results confirm the potential of urinary metabolomics for noninvasive diagnostic/prognostic screening and show that the antiviral nucleosides could represent novel biomarkers linking viral load, immune response, and metabolism. Moreover, we established for the first time a casual link between kynurenine accumulation and deranged NAD+/SIRT1, offering a novel mechanism through which SARS-CoV-2 manipulates host physiology.

Combined RNA interference and gene replacement therapy targeting MFN2 as proof of principle for the treatment of Charcot-Marie-Tooth type 2A.

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.

Chronic kidney disease-associated pruritus (CKD-aP) is associated with worse quality of life and increased healthcare utilization among dialysis patients.

PURPOSE: Chronic pruritus significantly impairs hemodialysis patients' health status and quality of life (QOL) and it is associated with higher mortality rate, more frequent hospitalizations, poorer dialysis and medication adherence, and deteriorated mental status. However, pruritus is still underestimated, underdiagnosed, and undertreated in the real-life clinical scenario. We investigated prevalence, clinical characteristics, clinical correlates, severity as well as physical and psychological burden of chronic pruritus among adult hemodialysis patients in a large international real-world cohort. METHODS: We conducted a retrospective cross-sectional study of patients registered in 152 Fresenius Medical Care (FMC) NephroCare clinics located in Italy, France, Ireland, United Kingdom, and Spain. Demographic and medical data were retrieved from the EuCliD® (European Clinical) database, while information on pruritus and QoL were abstracted from KDQOL™-36 and 5-D Itch questionnaire scores. RESULTS: A total of 6221 patients were included, of which 1238 were from France, 163 Ireland, 1469 Italy, 2633 Spain, and 718 UK. The prevalence of mild-to-severe pruritus was 47.9% (n = 2977 patients). Increased pruritus severity was associated with increased use of antidepressants, antihistamines, and gabapentin. Patients with severe pruritus more likely suffered from diabetes, more frequently missed dialysis sessions, and underwent more hospitalizations due to infections. Both mental and physical QOL scores were progressively lower as the severity of pruritus increased; this association was robust to adjustment for potential confounders. CONCLUSION: This international real-world analysis confirms that chronic pruritus is a highly prevalent condition among dialysis patients and highlights its considerable burden on several dimensions of patients' life.

Clinical assessment of liver metabolism during hypothermic oxygenated machine perfusion using microdialysis.

BACKGROUND: While growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood. METHODS: To assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome. RESULTS: Median (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2nd-hour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN. These correlations were not apparent when perfusate levels were considered. In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts. CONCLUSION: MD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment.

Addition of 5% CO2 to Inspiratory Gas Prevents Lung Injury in an Experimental Model of Pulmonary Artery Ligation.

Rationale: Unilateral ligation of the pulmonary artery may induce lung injury through multiple mechanisms, which might be dampened by inhaled CO2. Objectives: This study aims to characterize bilateral lung injury owing to unilateral ligation of the pulmonary artery in healthy swine undergoing controlled mechanical ventilation and its prevention by 5% CO2 inhalation and to investigate relevant pathophysiological mechanisms. Methods: Sixteen healthy pigs were allocated to surgical ligation of the left pulmonary artery (ligation group), seven to surgical ligation of the left pulmonary artery and inhalation of 5% CO2 (ligation + FiCO2 5%), and six to no intervention (no ligation). Then, all animals received mechanical ventilation with Vt 10 ml/kg, positive end-expiratory pressure 5 cm H2O, respiratory rate 25 breaths/min, and FiO2 50% (±FiCO2 5%) for 48 hours or until development of severe lung injury. Measurements and Main Results: Histological, physiological, and quantitative computed tomography scan data were compared between groups to characterize lung injury. Electrical impedance tomography and immunohistochemistry analysis were performed in a subset of animals to explore mechanisms of injury. Animals from the ligation group developed bilateral lung injury as assessed by significantly higher histological score, larger increase in lung weight, poorer oxygenation, and worse respiratory mechanics compared with the ligation + FiCO2 5% group. In the ligation group, the right lung received a larger fraction of Vt and inflammation was more represented, whereas CO2 dampened both processes. Conclusions: Mechanical ventilation induces bilateral lung injury within 48 hours in healthy pigs undergoing left pulmonary artery ligation. Inhalation of 5% CO2 prevents injury, likely through decreased stress to the right lung and antiinflammatory effects.

Viability testing during liver preservation.

PURPOSE OF REVIEW: Viability assessment is one of the main indications for machine perfusion (MP) in liver transplantation. This review summarizes the rationale, evolution and limitations of proposed viability criteria and suggests a framework for future studies. RECENT FINDINGS: Liver viability is most frequently assessed during normothermic MP by combining parameters relative to perfusate and bile composition, vascular flows and macroscopic aspect. Assessment protocols are largely heterogeneous and have significantly evolved over time, also within the same group, reflecting the ongoing evolution of the subject. Several recent preclinical studies using discarded human livers or animal models have explored other approaches to viability assessment. During hypothermic MP, perfusate flavin mononucleotide has emerged as a promising biomarker of mitochondrial injury and function. Most studies on the subject suffer from limitations, including low numbers, lack of multicenter validation, and subjective interpretation of some viability parameters. SUMMARY: MP adds a further element of complexity in the process of assessing the quality of a liver graft. Understanding the physiology of the parameters included in the different assessment protocols is necessary for their correct interpretation. Despite the possibility of assessing liver viability during MP, the importance of donor-recipient matching and operational variables should not be disregarded.

Sequential Use of Normothermic Regional and Ex Situ Machine Perfusion in Donation After Circulatory Death Liver Transplant.

In Italy, 20 minutes of a continuous flat line on an electrocardiogram are required for declaration of death. In the setting of donation after circulatory death (DCD), prolonged warm ischemia time prompted the introduction of abdominal normothermic regional perfusion (NRP) followed by postprocurement ex situ machine perfusion (MP). This is a retrospective review of DCD liver transplantations (LTs) performed at 2 centers using sequential NRP and ex situ MP. From January 2018 to April 2019, 34 DCD donors were evaluated. Three (8.8%) were discarded before NRP, and 11 (32.4%) were discarded based on NRP parameters (n = 1, 3.0%), liver macroscopic appearance at procurement and/or biopsy results (n = 9, 26.5%), or severe macroangiopathy at back-table evaluation (n = 1, 3.0%). A total of 20 grafts (58.8%; 11 uncontrolled DCDs, 9 controlled DCDs) were considered eligible for LT, procured and perfused ex situ (9 normothermic and 11 dual hypothermic MPs). In total, 18 (52.9%; 11 uncontrolled) livers were eventually transplanted. Median (interquartile range) no-flow time was 32.5 (30-39) minutes, whereas median functional warm ischemia time was 52.5 (47-74) minutes (controlled DCD), and median low-flow time was 112 minutes (105-129 minutes; uncontrolled DCD). There was no primary nonfunction, while postreperfusion syndrome occurred in 8 (44%) recipients. Early allograft dysfunction happened in 5 (28%) patients, while acute kidney injury occurred in 5 (28%). After a median follow-up of 15.1 (9.5-22.3) months, 1 case of ischemic-type biliary lesions and 1 patient death were reported. DCD LT is feasible even with the 20-minute no-touch rule. Strict NRP and ex situ MP selection criteria are needed to optimize postoperative results.

The Role of Ex Situ Hypothermic Oxygenated Machine Perfusion and Cold Preservation Time in Extended Criteria Donation After Circulatory Death and Donation After Brain Death.

Hypothermic oxygenated machine perfusion (HOPE) has the potential to counterbalance the detrimental consequences of cold and warm ischemia time (WIT) in both donation after brain death (DBD) and donation after circulatory death (DCD). Herein we investigated the protective effects of HOPE in extended criteria donor (ECD) DBD and overextended WIT DCD grafts. The present retrospective case series included 50 livers subjected to end-ischemic HOPE or dual DHOPE in 2 liver transplantation (LT) centers from January 2018 to December 2019. All DCD donors were subjected to normothermic regional perfusion before organ procurement. Results are expressed as median (interquartile range [IQR]). In the study period, 21 grafts were derived from overextended WIT DCD donors (total WIT 54 [IQR, 40-60] minutes and 75% classified as futile), whereas 29 were from ECD DBD. A total of 3 biliary complications and 1 case of ischemia-type biliary lesion were diagnosed. The rate of early allograft dysfunction (EAD) was 20%, and those patients had higher Comprehensive Complication Index scores. Through a changing point analysis, cold preservation time >9 hours was associated with prolonged hospital stays (P = 0.02), higher rates of EAD (P = 0.009), and worst post-LT complications (P = 0.02). Logistic regression analyses indicated a significant relationship between cold preservation time and EAD. No differences were shown in terms of the early post-LT results between LTs performed with DCD and DBD. Overall, our data are fully comparable with benchmark criteria in LT. In conclusion, the application of DHOPE obtained satisfactory and promising results using ECD-DBD and overextended DCD grafts. Our findings indicate the need to reduce cold preservation time also in the setting of DHOPE, particularly for grafts showing poor quality.

Physicians' prescribing behaviour and clinical practice patterns for allergic rhinitis management in Italy.

BACKGROUND: Despite availability of clinical guidelines, underdiagnosis, undertreatment, and poor adherence are still significant concerns in allergic rhinitis (AR) therapeutic management. We investigated clinical practice patterns and prescribing behavior of Italian healthcare professionals (HCPs) specialized in AR. METHODS: One-hundred allergologists, 100 ear, nose and throat (ENT) specialists, and 150 general practitioners (GPs) were recruited. The survey assessed: socio-demographic, work experience, monthly caseload, prescription drivers. Next, HCPs were invited to retrospectively recover patients' clinical data to investigate: AR clinical characteristics, therapy management, prescription patterns, patient adherence. Descriptive statistics, Chi square, One-Way analysis of variance, and Two-Way Analysis of Variance were performed. RESULTS: Allergologists visited more AR patients (31% of monthly caseload) than ENTs (21%, p < 0.001), while GPs' caseload was the lowest (6%). Clinical information of 2823 patients were retrieved of whom 1906 (67.5%) suffered from moderate/severe AR (discomfort score: 7.7 ± 1.3) and 917 (32.4%) from mild AR (5.7 ± 1.9). About one-third of mild patients had a discomfort score ≥ 7. Main prescription drivers were "effective on all symptoms" (54.3% patients) and "quick symptom relief" (47.8%), whereas minor drivers were "affordable price" (13.4%) and "refundable" (8.7%). The most prescribed drugs were antihistamines and intranasal corticosteroids (79% and 55% prescriptions), followed by fixed-dose-combination of intranasal azelastine/fluticasone (19%). Polytherapy was the most common treatment strategy (59.6%). HCPs' believe that the majority of the patients was adherent to treatment (88% with score > 7). CONCLUSIONS: This survey describes the therapeutic approach adopted by Italian physicians to cope with AR and shows that HCPs underestimated AR severity and had a non-realistic perception of patients' adherence. These findings suggest that further efforts are required to improve AR clinical management in Italy.

Information Needs and Information-Seeking Behavior of Italian Neurologists: Exploratory Mixed Methods Study.

BACKGROUND: Current medical professions involve an extensive knowledge of the latest validated scientific data to implement disease diagnosis, therapeutic strategies, and patient care. Although clinicians can refer to a growing number and type of information sources to keep current with new scientific achievements, there are still various concerns about medical information validity, quality, and applicability into clinical practice. Novel strategies are required to identify physicians' real-life needs with the final aim to improve modern medical information delivery. OBJECTIVE: Our research used an innovative tool to collect real-time physician queries in order to investigate information needs and seeking behavior of Italian neurologists treating patients with multiple sclerosis (MS) and migraine. METHODS: The study was designed as an exploratory mixed methods (ie, qualitative and quantitative) study involving 15 consecutive days of observation. A total of 50 neurologists (n=25 MS and n=25 migraine specialists) were recruited. Data were collected using an instant messaging mobile app designed for this research. At each information-seeking event, moderators triggered a computer-assisted personal interview including both semistructured interview and close-ended questions. Interactions and physician queries collected using the mobile app were coded into emerging themes by content analysis. RESULTS: Neurologist queries were relevant to the following major themes: therapy management (36/50, 71%) and drug-related information (34/50, 67%), followed by diagnostic strategies and procedures (21/50, 42%). Quantitative analysis indicated online resources were preferentially used by clinicians (48/50, 96%) compared with offline sources (24/50, 47%). A multichannel approach, in which both online and offline sources were consulted to meet the same need, was adopted in 33% (65/198) of information-seeking events. Neurologists more likely retrieved information from online relative to offline channels (F=1.7; P=.01). MS specialists were 53% more likely to engage in one information-seeking event compared with migraine neurologists (risk ratio 1.54; 95% CI 1.16-2.05). MS specialists tended to be more interested in patient-related content than migraine clinicians (28% [7/25] vs 10% [2/25], P=.06), who conversely more likely sought information concerning therapy management (85% [21/25] vs 60% [15/25], P=.05). Compared with MS clinicians, migraine specialists had a harder time finding the required information, either looking at online or offline channels (F=12.5; P=.01) and less frequently used offline channels (30% [8/25] vs 60% [15/25] of information-seeking events, P=.02). When multiple sources needed to be consulted to retrieve an information item, a reduced satisfaction rate was observed both among migraine and MS specialists (single source vs multiple sources P=.003). CONCLUSIONS: This study provides a detailed description of real-life seeking behavior, educational needs, and information sources adopted by Italian MS and migraine neurologists. Neurologist information needs and seeking behavior reflect the specific characteristics of the specialty area in which they operate. These findings suggest identification of time- and context-specific needs of clinicians is required to design an effective medical information strategy.

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

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.

Effects of sodium citrate, citric acid and lactic acid on human blood coagulation.

INTRODUCTION: Citric acid infusion in extracorporeal blood may allow concurrent regional anticoagulation and enhancement of extracorporeal CO2 removal. Effects of citric acid on human blood thromboelastography and aggregometry have never been tested before. METHODS: In this in vitro study, citric acid, sodium citrate and lactic acid were added to venous blood from seven healthy donors, obtaining concentrations of 9 mEq/L, 12 mEq/L and 15 mEq/L. We measured gas analyses, ionized calcium (iCa++) concentration, activated clotting time (ACT), thromboelastography and multiplate aggregometry. Repeated measure analysis of variance was used to compare the acidifying and anticoagulant properties of the three compounds. RESULTS: Sodium citrate did not affect the blood gas analysis. Increasing doses of citric and lactic acid progressively reduced pH and HCO3- and increased pCO2 (p<0.001). Sodium citrate and citric acid similarly reduced iCa++, from 0.39 (0.36-0.39) and 0.35 (0.33-0.36) mmol/L, respectively, at 9 mEq/L to 0.20 (0.20-0.21) and 0.21 (0.20-0.23) mmol/L at 15 mEq/L (p<0.001). Lactic acid did not affect iCa++ (p=0.07). Sodium citrate and citric acid similarly incremented the ACT, from 234 (208-296) and 202 (178-238) sec, respectively, at 9 mEq/L, to >600 sec at 15 mEq/L (p<0.001). Lactic acid did not affect the ACT values (p=0.486). Sodium citrate and citric acid similarly incremented R-time and reduced α-angle and maximum amplitude (MA) (p<0.001), leading to flat-line thromboelastograms at 15 mEq/L. Platelet aggregometry was not altered by any of the three compounds. CONCLUSIONS: Citric acid infusions determine acidification and anticoagulation of blood similar to lactic acid and sodium citrate, respectively.

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

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.

α-Melanocyte-stimulating-hormone (α-MSH) modulates human chondrocyte activation induced by proinflammatory cytokines.

BACKGROUND: Alpha-melanocyte-stimulating-hormone (α-MSH) has marked anti-inflammatory potential. Proinflammatory cytokines are critical mediators of the disturbed cartilage homeostasis in osteoarthritis, inhibiting anabolic activities and increasing catabolic activities in chondrocytes. Since human chondrocytes express α-MSH receptors, we evaluated the role of the peptide in modulating chondrocyte production of pro-inflammatory cytokines, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in response to interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). METHODS: Human articular chondrocytes were obtained from osteoarthritic joint cartilage from subjects undergoing hip routine arthroplasty procedures. The cells were cultured with or without α-MSH in the presence of IL-1ß or TNF-α. Cell-free supernatants were collected and cells immediately lysed for RNA purification. Expression of cytokines, MMPs, TIMPs, iNOS was determined by Reverse Transcription Real-time Polymerase Chain Reaction and enzyme-linked immunosorbent assay. Griess reaction was used for NO quantification. RESULTS: Gene expression and secretion of IL-6, IL-8, MMP-3, MMP-13 were significantly increased in IL-1ß or TNF-α-stimulated chondrocytes; α-MSH did not modify the release of IL-6 or IL-8 while the peptide significantly reduced their gene expression on TNF-α-stimulated cells. A significant inhibition of MMP3 gene expression and secretion from IL-1ß or TNFα-stimulated chondrocytes was induced by α-MSH. On the other hand, α-MSH did not modify the release of MMP-13 by cytokine-stimulated chondrocyte but significantly decreased gene expression of the molecule on TNF-α-stimulated cells. Detectable amount of TIMP-3 and TIMP-4 were present in the supernatants of resting chondrocytes and a significant increase of TIMP-3 gene expression and release was induced by α-MSH on unstimulated cells. TIMP-3 secretion and gene expression were significantly increased in IL-1ß-stimulated chondrocytes and α-MSH down-regulated gene expression but not secretion of the molecule. TIMP-4 gene expression (but not secretion) was moderately induced in IL-1ß-stimulated chondrocytes with a down-regulation exerted by α-MSH. IL-1ß and TNF-α were potent stimuli for NO production and iNOS gene expression by chondrocytes; no inhibition was induced by α-MSH on cytokine-stimulated NO production, while the peptide significantly reduced gene expression of iNOS. CONCLUSIONS: Our results underscore a potential anti-inflammatory and chondroprotective activity exerted by α-MSH, increasing TIMP-3 gene expression and release on resting cells and down- modulating TNF-α-induced activation of human chondrocytes. However, the discrepancy between the influences exerted by α-MSH on gene expression and protein release as well as the difference in the inhibitory pattern exerted by α-MSH in TNF-α- or IL-1ß-stimulated cells leave some uncertainty on the role of the peptide on chondrocyte modulation.

Twelve-hour normothermic liver perfusion in a rat model: characterization of the changes in the ex-situ bio-molecular phenotype and metabolism.

The partial understanding of the biological events that occur during normothermic machine perfusion (NMP) and particularly during prolonged perfusion might hinder its deployment in clinical transplantation. The aim of our study was to implement a rat model of prolonged NMP to characterize the bio-molecular phenotype and metabolism of the perfused organs. Livers (n = 5/group) were procured and underwent 4 h (NMP4h) or 12 h (NMP12h) NMP, respectively, using a perfusion fluid supplemented with an acellular oxygen carrier. Organs that were not exposed to any procedure served as controls (Native). All perfused organs met clinically derived viability criteria at the end of NMP. Factors related to stress-response and survival were increased after prolonged perfusion. No signs of oxidative damage were detected in both NMP groups. Evaluation of metabolite profiles showed preserved mitochondrial function, activation of Cori cycle, induction of lipolysis, acetogenesis and ketogenesis in livers exposed to 12 h-NMP. Increased concentrations of metabolites involved in glycogen synthesis, glucuronidation, bile acid conjugation, and antioxidant response were likewise observed. In conclusion, our NMP12h model was able to sustain liver viability and function, thereby deeply changing cell homeostasis to maintain a newly developed equilibrium. Our findings provide valuable information for the implementation of optimized protocols for prolonged NMP.

Brain-Derived Neurotrophic Factor-Loaded Low-Temperature-Sensitive liposomes as a drug delivery system for repairing podocyte damage.

Podocytes, cells of the glomerular filtration barrier, play a crucial role in kidney diseases and are gaining attention as potential targets for new therapies. Brain-Derived Neurotrophic Factor (BDNF) has shown promising results in repairing podocyte damage, but its efficacy via parenteral administration is limited by a short half-life. Low temperature sensitive liposomes (LTSL) are a promising tool for targeted BDNF delivery, preserving its activity after encapsulation. This study aimed to improve LTSL design for efficient BDNF encapsulation and targeted release to podocytes, while maintaining stability and biological activity, and exploiting the conjugation of targeting peptides. While cyclic RGD (cRGD) was used for targeting endothelial cells in vitro, a homing peptide (HITSLLS) was conjugated for more specific uptake by glomerular endothelial cells in vivo. BDNF-loaded LTSL successfully repaired cytoskeleton damage in podocytes and reduced albumin permeability in a glomerular co-culture model. cRGD conjugation enhanced endothelial cell targeting and uptake, highlighting an improved therapeutic effect when BDNF release was induced by thermoresponsive liposomal degradation. In vivo, targeted LTSL showed evidence of accumulation in the kidneys, and their BDNF delivery decreased proteinuria and ameliorated kidney histology. These findings highlight the potential of BDNF-LTSL formulations in restoring podocyte function and treating glomerular diseases.

Pathophysiological profile of non-ventilated lung injury in healthy female pigs undergoing mechanical ventilation.

BACKGROUND: Lung regions excluded from mechanical insufflation are traditionally assumed to be spared from ventilation-associated lung injury. However, preliminary data showed activation of potential mechanisms of injury within these non-ventilated regions (e.g., hypoperfusion, inflammation). METHODS: In the present study, we hypothesized that non-ventilated lung injury (NVLI) may develop within 24 h of unilateral mechanical ventilation in previously healthy pigs, and we performed extended pathophysiological measures to profile NVLI. We included two experimental groups undergoing exclusion of the left lung from the ventilation with two different tidal volumes (15 vs 7.5 ml/kg) and a control group on bilateral ventilation. Pathophysiological alteration including lung collapse, changes in lung perfusion, lung stress and inflammation were measured. Lung injury was quantified by histological score. RESULTS: Histological injury score of the non-ventilated lung is significantly higher than normally expanded lung from control animals. The histological score showed lower intermediate values (but still higher than controls) when the tidal volume distending the ventilated lung was reduced by 50%. Main pathophysiological alterations associated with NVLI were: extensive lung collapse; very low pulmonary perfusion; high inspiratory airways pressure; and higher concentrations of acute-phase inflammatory cytokines IL-6, IL-1ß and TNF-α and of Angiopoietin-2 (a marker of endothelial activation) in the broncho-alveolar lavage. Only the last two alterations were mitigated by reducing tidal volume, potentially explaining partial protection. CONCLUSIONS: Non-ventilated lung injury develops within 24 h of controlled mechanical ventilation due to multiple pathophysiological alterations, which are only partially prevented by low tidal volume.

Respiratory failure that occurs in cases of atelectasis, pneumonia and acute hypoxemic respiratory failure a machine called a mechanical ventilator is used to move air in and out of the patient's lungs. We know that the use of a mechanical ventilator can induce lung injury, but complete exclusion from ventilation might not be safe. Using pig lungs to mimic the patient's lungs, we evaluated the use of a ventilator against non-use. We find that the lungs sustained injury regardless of ventilator use. The non-ventilated lung injury consisted of collapse (lack of expansion), low amount of blood flow, high ventilation pressure and inflammatory response. Physicians should be aware that also the regions of the lung not receiving ventilation are at risk of injury.

Portal hypertension-like pattern in coronavirus disease 2019 acute respiratory distress syndrome.

OBJECTIVES: Although respiratory failure is the most common feature in coronavirus disease 2019 (COVID-19), abdominal organ involvement is likewise frequently observed. To investigate visceral and thoracic circulation and abdominal organ damage in COVID-19 patients. MATERIALS AND METHODS: A monocentric observational study was carried on. In COVID-19 patients affected by acute respiratory distress syndrome (ARDS) (n = 31) or mild pneumonia (n = 60) thoracoabdominal circulation was evaluated using Doppler-ultrasound and computed tomography. The study also included non-COVID-19 patients affected by ARDS (n = 10) or portal hypertension (n = 10) for comparison of the main circulatory changes. RESULTS: Patients affected by COVID-19 ARDS showed hyperdynamic visceral flow and increased portal velocity, hepatic artery resistance-index, and spleen diameter relative to those with mild-pneumonia (p = 0.001). Splanchnic circulatory parameters significantly correlated with the main respiratory indexes (p < 0.001) and pulmonary artery diameter (p = 0.02). The chest and abdominal vascular remodeling pattern of COVID-19 ARDS patients resembled the picture observed in the PH group, while differed from that of the non-COVID ARDS group. A more severe COVID-19 presentation was associated with worse liver dysfunction and enhanced inflammatory activation; these parameters both correlated with abdominal (p = 0.04) and chest imaging measures (p = 0.03). CONCLUSION: In COVID-19 ARDS patients there are abdominal and lung vascular modifications that depict a portal hypertension-like pattern. The correlation between visceral vascular remodeling, pulmonary artery enlargement, and organ damage in these critically ill patients is consistent with a portal hyperlfow-like syndrome that could contribute to the peculiar characteristics of respiratory failure in these patients. CLINICAL RELEVANCE STATEMENT: our data suggest that the severity of COVID-19 lung involvement is directly related to the development of a portal hyperflow-like syndrome. These observations should help in defining the need for a closer monitoring, but also to develop dedicated therapeutic strategies.

Microbiota dysbiosis influences immune system and muscle pathophysiology of dystrophin-deficient mice.

Duchenne muscular dystrophy (DMD) is a progressive severe muscle-wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ-free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.