ABSTRACT
Tidal volume (TV) monitoring breath-by-breath is not available at bedside in non-intubated patients. However, TV monitoring may be useful to evaluate the work of breathing. A non-invasive device based on bioimpedance provides continuous and real-time volumetric tidal estimation during spontaneous breathing. We performed a prospective study in healthy volunteers aimed at evaluating the accuracy, the precision and the trending ability of measurements of ExSpiron®Xi as compared with the gold standard (i.e. spirometry). Further, we explored whether the differences between the 2 devices would be improved by the calibration of ExSpiron®Xi with a pre-determined tidal volume. Analysis accounted for the repeated nature of measurements within each subject. We enrolled 13 healthy volunteers, including 5 men and 8 women. Tidal volume, TV/ideal body weight (IBW) and respiratory rate (RR) measured with spirometer (TVSpirometer) and with ExSpiron®Xi (TVExSpiron) showed a robust correlation, while minute ventilation (MV) showed a weak correlation, in both non/calibrated and calibrated steps. The analysis of the agreement showed that non-calibrated TVExSpiron underestimated TVspirometer, while in the calibrated steps, TVExSpiron overestimated TVspirometer. The calibration procedure did not reduce the average absolute difference (error) between TVSpirometer and TVExSpiron. This happened similarly for TV/IBW and MV, while RR showed high accuracy and precision. The trending ability was excellent for TV, TV/IBW and RR. The concordance rate (CR) was >95% in both calibrated and non-calibrated measurements. The trending ability of minute ventilation was limited. Absolute error for both calibrated and not calibrated values of TV, TV/IBW and MV accounting for repeated measurements was variably associated with BMI, height and smoking status. Conclusions: Non-invasive TV, TV/IBW and RR estimation by ExSpiron®Xi was strongly correlated with tidal ventilation according to the gold standard spirometer technique. This data was not confirmed for MV. The calibration of the device did not improve its performance. Although the accuracy of ExSpiron®Xi was mild and the precision was limited for TV, TV/IBW and MV, the trending ability of the device was strong specifically for TV, TV/IBW and RR. This makes ExSpiron®Xi a non-invasive monitoring system that may detect real-time tidal volume ventilation changes and then suggest the need to better optimize the patient ventilatory support.
Subject(s)
Respiration , Male , Humans , Female , Prospective Studies , Healthy Volunteers , Tidal Volume , Lung Volume Measurements/methodsABSTRACT
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 drm1drm2 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 spl/nzz 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 ago9, rdr6 and drm1drm2 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, SEEDSTICK, directly regulates AGO9 and RDR6 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.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , DNA Methylation/genetics , MADS Domain Proteins/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Arabidopsis/growth & development , Argonaute Proteins/genetics , Gene Expression Regulation, Plant/genetics , Germ Cells/growth & development , Germ Cells/metabolism , Methyltransferases/genetics , Mutation/genetics , Ovule/genetics , Plant Development/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , RNA/genetics , RNA-Dependent RNA Polymerase/genetics , Stem Cells/cytologyABSTRACT
OBJECTIVES: Extracorporeal carbon dioxide removal is used to treat patients suffering from acute respiratory failure. However, the procedure is hampered by the high blood flow required to achieve a significant CO2 clearance. We aimed to develop an ultralow blood flow device to effectively remove CO2 combined with continuous renal replacement therapy (CRRT). DESIGN: Preclinical, proof-of-concept study. SETTING: An extracorporeal circuit where 200 mL/min of blood flowed through a hemofilter connected to a closed-loop dialysate circuit. An ion-exchange resin acidified the dialysate upstream, a membrane lung to increase Pco2 and promote CO2 removal. PATIENTS: Six, 38.7 ± 2.0-kg female pigs. INTERVENTIONS: Different levels of acidification were tested (from 0 to 5 mEq/min). Two l/hr of postdilution CRRT were performed continuously. The respiratory rate was modified at each step to maintain arterial Pco2 at 50 mm Hg. MEASUREMENTS AND MAIN RESULTS: Increasing acidification enhanced CO2 removal efficiency of the membrane lung from 30 ± 5 (0 mEq/min) up to 145 ± 8 mL/min (5 mEq/min), with a 483% increase, representing the 73% ± 7% of the total body CO2 production. Minute ventilation decreased accordingly from 6.5 ± 0.7 to 1.7 ± 0.5 L/min. No major side effects occurred, except for transient tachycardia episodes. As expected from the alveolar gas equation, the natural lung Pao2 dropped at increasing acidification steps, given the high dissociation between the oxygenation and CO2 removal capability of the device, thus Pao2 decreased. CONCLUSIONS: This new extracorporeal ion-exchange resin-based multiple-organ support device proved extremely high efficiency in CO2 removal and continuous renal support in a preclinical setting. Further studies are required before clinical implementation.
Subject(s)
Continuous Renal Replacement Therapy , Animals , Carbon Dioxide , Dialysis Solutions , Female , Humans , Oxygen , Respiration, Artificial/methods , SwineABSTRACT
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.
Subject(s)
Carbon Dioxide/therapeutic use , Disease Models, Animal , Lung Injury/prevention & control , Protective Agents/therapeutic use , Pulmonary Artery/surgery , Respiration, Artificial/adverse effects , Swine/surgery , Administration, Inhalation , Animals , Female , Ligation , Lung Injury/etiology , Lung Injury/physiopathology , Lung Injury/therapy , Treatment OutcomeABSTRACT
Rationale: 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.Objectives: To evaluate feasibility, safety, and effectiveness of a novel strategy to correct acidemia through extracorporeal chloride removal by electrodialysis.Methods: 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.Measurements and Main Results: 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; P < 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; P < 0.001). No complications occurred.Conclusions: We described the first in vivo 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.
Subject(s)
Acidosis/blood , Acidosis/therapy , Chlorides/blood , Renal Dialysis/methods , Animals , Electricity , Extracorporeal Circulation , SwineABSTRACT
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 A1c (HbA1c) 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., ß-cell function, insulin clearance and insulin sensitivity, and diabetes regression after OLT. METHODS: Eighty cirrhotic patients with non-diabetic FPG and HbA1c levels underwent an extended oral glucose tolerance test (OGTT) before and 3, 6, 12 and 24â¯months after OLT. The OGTT data were analysed with a mathematical model to estimate derivative control (DC) and proportional control (PC) of ß-cell function and insulin clearance (which determine insulin bioavailability), and with the Oral Glucose Insulin Sensitivity (OGIS)-2â¯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-2â¯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 ß-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 ß-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 ß-cell function in these individuals. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02038517.
Subject(s)
Diabetes Mellitus/physiopathology , Insulin-Secreting Cells/physiology , Liver Cirrhosis/surgery , Liver Transplantation , Adult , Aged , Blood Glucose/analysis , Female , Glycated Hemoglobin/analysis , Humans , Liver Cirrhosis/physiopathology , Longitudinal Studies , Male , Middle AgedABSTRACT
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.
Subject(s)
Apoptosis , Heat-Shock Response , Lung/metabolism , MAP Kinase Signaling System , Oxidative Stress , Perfusion , Animals , Cell Survival , Extracellular Signal-Regulated MAP Kinases/metabolism , Hyaluronan Synthases/metabolism , Hyaluronic Acid/metabolism , Male , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Patient Outcome Assessment , Positive-Pressure Respiration/mortality , Positive-Pressure Respiration/methods , Respiratory Distress Syndrome/mortality , Respiratory Distress Syndrome/therapy , Aged , Cohort Studies , Female , Humans , Lung/diagnostic imaging , Lung/physiopathology , Lung Compliance , Male , Middle Aged , Retrospective Studies , Tidal Volume , Tomography, X-Ray ComputedABSTRACT
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 (TR1) cells but is also produced by CD25+ regulatory T (Treg) cells. OBJECTIVE: We aimed to identify and characterize human intestinal TR1 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-1ß and IL-23 responsiveness was assessed. RESULTS: Intestinal TR1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5+PD-1+ TR1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ+ TR1 cells, but not IL-7 receptor-positive TH cells or CD25+ Treg cells, showed lower IL-10 expression in patients with IBDs. TR1 cells were responsive to IL-23, and IFN-γ+ TR1 cells downregulated IL-10 with IL-1ß 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 TR1 cells. Selective downregulation of IL-10 by IFN-γ+ TR1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.
Subject(s)
Cytokines/immunology , Inflammatory Bowel Diseases/immunology , Intestinal Mucosa/immunology , Programmed Cell Death 1 Receptor/immunology , Receptors, CCR5/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Aged , Animals , Cells, Cultured , Colonic Neoplasms/immunology , Female , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Young AdultABSTRACT
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.
Subject(s)
Biomarkers/metabolism , Lung/metabolism , Metabolic Networks and Pathways , Proteomics/methods , Animals , Chromatography, Liquid , Models, Animal , Nanotechnology , Organ Preservation , Perfusion , Protein Interaction Maps , Rats , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Amino Acid Oxidoreductases/biosynthesis , Insulin Resistance , Liver Cirrhosis/enzymology , Liver/enzymology , Non-alcoholic Fatty Liver Disease/enzymology , Animals , Cell Proliferation , Cell Transdifferentiation , Cells, Cultured , Choline Deficiency/complications , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Enzyme Induction , Extracellular Matrix/metabolism , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolism , Genetic Predisposition to Disease , Hepatic Stellate Cells/enzymology , Hepatic Stellate Cells/pathology , Hepatocytes/enzymology , Hepatocytes/pathology , Humans , Liver/pathology , Liver Cirrhosis/etiology , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Methionine/deficiency , Mice, Inbred C57BL , Mice, Knockout , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/pathology , Phenotype , Receptor, Insulin/deficiency , Receptor, Insulin/genetics , Signal TransductionABSTRACT
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.
Subject(s)
Immunoproteins/antagonists & inhibitors , Muscular Dystrophy, Duchenne/drug therapy , Proteasome Inhibitors/administration & dosage , T-Lymphocytes/immunology , Animals , Cell Differentiation , Disease Models, Animal , Genetic Therapy , Mice , Mice, Inbred mdx , Muscular Dystrophy, Duchenne/immunology , Oligopeptides/administration & dosage , Oligopeptides/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/physiologyABSTRACT
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.
Subject(s)
Cardiomegaly/metabolism , MAP Kinase Signaling System/drug effects , Niacinamide/analogs & derivatives , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/metabolism , Animals , Cardiomegaly/diagnosis , Cardiomegaly/drug therapy , Cardiomegaly/genetics , Cell Line , Cytoskeleton/metabolism , Disease Models, Animal , Extracellular Matrix/metabolism , Gap Junctions/metabolism , Gene Expression Regulation , Heart Ventricles/metabolism , Heart Ventricles/pathology , Mice , Mice, Transgenic , Niacinamide/pharmacology , Phenotype , Proto-Oncogene Proteins c-met/genetics , Ventricular Remodeling/geneticsABSTRACT
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.
Subject(s)
Pulmonary Edema/etiology , Respiration, Artificial/adverse effects , Respiratory Mechanics/physiology , Ventilator-Induced Lung Injury/etiology , Animals , Functional Residual Capacity/physiology , Humans , Lung Compliance/physiology , Pulmonary Edema/physiopathology , Stress, Mechanical , Swine , Tidal Volume/physiology , Ventilator-Induced Lung Injury/physiopathologyABSTRACT
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.
Subject(s)
Ventilator-Induced Lung Injury/physiopathology , Ventilators, Mechanical , Air Pressure , Animals , Elasticity , Equipment Design , Inspiratory Capacity , Lung/diagnostic imaging , Lung/pathology , Lung/physiopathology , Mechanical Phenomena , Organ Size , Pulmonary Edema/chemically induced , Pulmonary Edema/pathology , Radiography , Respiratory Rate , Sus scrofa , Ventilator-Induced Lung Injury/pathologyABSTRACT
Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease.
ABSTRACT
BACKGROUND & AIMS: This study evaluated the contribution of ß-cell dysfunction and insulin resistance to cirrhosis-associated diabetes. METHODS: One-hundred and sixty cirrhotic patients with normal fasting plasma glucose (FPG), three with impaired fasting glucose and seven with untreated diabetes mellitus (DM) underwent an extended oral glucose tolerance test (OGTT). The OGTT data were analyzed with a Minimal Model to estimate dynamic (derivative) control (DC) and static (proportional) control (PC) of ß-cell function, and with the Oral Glucose Insulin Sensitivity (OGIS)-2h index to estimate insulin sensitivity. RESULTS: Twenty-six patients (15.6%) had normal glucose tolerance (NGT), 60 (35.8%) had impaired glucose tolerance (IGT), and 84 (48.6%) had DM. DC was significantly reduced in DM vs. NGT and IGT patients. PC was significantly impaired in DM and IGT vs. NGT patients and in DM vs. IGT subjects. The OGIS-2h index was significantly reduced to a similar extent in DM and IGT vs. NGT patients. Patients with Child-Pugh class B and C cirrhosis had reduced DC and PC, but not OGIS-2h values, as compared with subjects in class A. Moreover, Child-Pugh class/score was an independent predictor of ß-cell function even after adjustment for glucose tolerance. CONCLUSIONS: Abnormalities of glucose tolerance occur frequently in cirrhosis even in patients with normal FPG, thereby supporting the importance of performing an OGTT. Transition from IGT to DM is driven primarily by ß-cell dysfunction. Insulin secretion worsens in parallel with the severity of liver disease, thus suggesting a detrimental effect of liver failure on pancreatic islets on its own.
Subject(s)
Diabetes Complications/physiopathology , Insulin Resistance , Insulin-Secreting Cells/physiology , Liver Cirrhosis/complications , Blood Glucose/metabolism , Cross-Sectional Studies , Diabetes Complications/etiology , Female , Glucose Tolerance Test , Hepatitis C, Chronic/complications , Humans , Liver Cirrhosis/etiology , Liver Cirrhosis/physiopathology , Liver Transplantation , Male , Middle Aged , Severity of Illness IndexABSTRACT
Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor. The HGF/Met pathway has a prominent role in cardiovascular remodelling after tissue injury. The present review provides a synopsis of the cellular and molecular mechanisms underlying the effects of HGF/Met in the heart and blood vessels. In vivo, HGF/Met function is particularly important for the protection of the heart in response to both acute and chronic insults, including ischaemic injury and doxorubicin-induced cardiotoxicity. Accordingly, conditional deletion of Met in cardiomyocytes results in impaired organ defence against oxidative stress. After ischaemic injury, activation of Met provides strong anti-apoptotic stimuli for cardiomyocytes through PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase) cascades. Recently, we found that HGF/Met is also important for autophagy regulation in cardiomyocytes via the mTOR (mammalian target of rapamycin) pathway. HGF/Met induces proliferation and migration of endothelial cells through Rac1 (Ras-related C3 botulinum toxin substrate 1) activation. In fibroblasts, HGF/Met antagonizes the actions of TGFß1 (transforming growth factor ß1) and AngII (angiotensin II), thus preventing fibrosis. Moreover, HGF/Met influences the inflammatory response of macrophages and the immune response of dendritic cells, indicating its protective function against atherosclerotic and autoimmune diseases. The HGF/Met axis also plays an important role in regulating self-renewal and myocardial regeneration through the enhancement of cardiac progenitor cells. HGF/Met has beneficial effects against myocardial infarction and endothelial dysfunction: the cellular and molecular mechanisms underlying repair function in the heart and blood vessels are common and include pro-angiogenic, anti-inflammatory and anti-fibrotic actions. Thus administration of HGF or HGF mimetics may represent a promising therapeutic agent for the treatment of both coronary and peripheral artery disease.
Subject(s)
Blood Vessels/enzymology , Cardiovascular Diseases/enzymology , Hepatocyte Growth Factor/metabolism , Myocardium/enzymology , Proto-Oncogene Proteins c-met/metabolism , Signal Transduction , Vascular Remodeling , Ventricular Remodeling , Animals , Apoptosis , Autophagy , Blood Vessels/drug effects , Blood Vessels/immunology , Blood Vessels/pathology , Blood Vessels/physiopathology , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/genetics , Cardiovascular Diseases/immunology , Cardiovascular Diseases/pathology , Cardiovascular Diseases/physiopathology , Dendritic Cells/enzymology , Dendritic Cells/immunology , Fibrosis , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/therapeutic use , Humans , Immune Tolerance , Inflammation/enzymology , Inflammation/pathology , Inflammation/prevention & control , Molecular Targeted Therapy , Myocardium/immunology , Myocardium/pathology , Neovascularization, Physiologic , Oxidative Stress , Proto-Oncogene Proteins c-met/genetics , Regeneration , Signal Transduction/drug effects , Vascular Remodeling/drug effects , Ventricular Remodeling/drug effectsABSTRACT
BACKGROUND: During mechanical ventilation, stress and strain may be locally multiplied in an inhomogeneous lung. The authors investigated whether, in healthy lungs, during high pressure/volume ventilation, injury begins at the interface of naturally inhomogeneous structures as visceral pleura, bronchi, vessels, and alveoli. The authors wished also to characterize the nature of the lesions (collapse vs. consolidation). METHODS: Twelve piglets were ventilated with strain greater than 2.5 (tidal volume/end-expiratory lung volume) until whole lung edema developed. At least every 3 h, the authors acquired end-expiratory/end-inspiratory computed tomography scans to identify the site and the number of new lesions. Lung inhomogeneities and recruitability were quantified. RESULTS: The first new densities developed after 8.4 ± 6.3 h (mean ± SD), and their number increased exponentially up to 15 ± 12 h. Afterward, they merged into full lung edema. A median of 61% (interquartile range, 57 to 76) of the lesions appeared in subpleural regions, 19% (interquartile range, 11 to 23) were peribronchial, and 19% (interquartile range, 6 to 25) were parenchymal (P < 0.0001). All the new densities were fully recruitable. Lung elastance and gas exchange deteriorated significantly after 18 ± 11 h, whereas lung edema developed after 20 ± 11 h. CONCLUSIONS: Most of the computed tomography scan new densities developed in nonhomogeneous lung regions. The damage in this model was primarily located in the interstitial space, causing alveolar collapse and consequent high recruitability.
Subject(s)
Lung/pathology , Respiration, Artificial/adverse effects , Ventilator-Induced Lung Injury/etiology , Ventilator-Induced Lung Injury/pathology , Ventilators, Mechanical/adverse effects , Animals , Animals, Newborn , Female , Respiration, Artificial/trends , Swine , Time Factors , Ventilators, Mechanical/trendsABSTRACT
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.