Nuclear magnetic resonance-based metabolomics in respiratory medicine

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DEVELOPMENT AND APPLICATION OF ADVANCED NMR TECHNIQUES FOR BIOMARKERS IDENTIFICATION

Biomarkers identification represents one of the most attractive goals in all biological research field due to its important application to improve diagnosis, guide molecularly targeted therapy and monitor activity and therapeutic response across a wide spectrum of disease and pathological states. With its ability to discover new metabolic markers, NMR metabolic profiling (metabonomics), coupled with pattern recognition methods, represents a highly effective approach for disease development and characterization. The purpose of the present thesis is to explore the recent NMR improvements by applying and developing new metabolomic strategies for biomarkers discovery, including NMR data handling, peaks quantification and fast data acquisition. We applied NMR spectroscopy and regression techniques to different patient classes to discriminate a) the progressive liver alterations during tumorigenesis and b) the exhaled breath condensate (EBC) of patients with airway diseases. In the first application a) Principal component analysis (PCA) and Orthogonal Projection to Latent Structures Discriminant Analysis (O2PLS-DA) showed that the disease evolution clearly followed the increase of the lactate together with the remarkable decrease of the glucose signal, thus suggesting that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions. In particular, we identified a statistical model that could be used to distinguish hepatic metastasis and human hepatocarcinoma from a "normal" (healthy) hepatic tissue. In the second application b) we investigated the role of pre-analytical variables (saliva and disinfectant contamination), potentially influencing EBC, to evaluate the stability and reproducibility of samples and to discriminate healthy subjects from patients with airway disease. Our results show that saliva does not hamper EBC discrimination of different samples, while the disinfectant could act as a strong contaminant. However, by selecting specific non-contaminated regions of spectra, Projection to Latent Structures-Discriminant Analysis (PLS-DA) was able to discriminate EBC of healthy subjects, laryngectomized and patients with chronic obstructive pulmonary disease (COPD). As a further enhanced tool for high thoughput NMR analysis, we developed and tested a new integration method for 2D NMR spectra quantification, called CAKE, based upon NMR axial symmetry and Monte Carlo Hit-or-Miss technique. Integration tests on simulated and experimental peaks with different degree of overlap, showed the CAKE efficacy in estimating umbiased peak volume, even for strongly overlapping peaks. Moreover, it is substantially independent on digital resolution and SNR. Finally, we successfully investigated the possibility of exploiting enhanced NMR pulse sequences for fast spectra acquisition. In particular, we applied the so-called SOFAST-HMQC pulse scheme to detect in-cell metabolism. we have applied the SOFAST experiment to 15N-labeled Thalassiosira rotula diatom cells obtaining, to the best of our knowledge, the first application of fast NMR spectroscopy. We collected spectra in 10-15 s of acquisition time, pinpointing the T. rotula 1H-15N metabolic profiling directly in living cells

Metabolomics of COPD Pulmonary Rehabilitation Outcomes via Exhaled Breath Condensate

: Chronic obstructive pulmonary disease (COPD) is characterized by different phenotypes and clinical presentations. Therefore, a single strategy of pulmonary rehabilitation (PR) does not always yield the expected clinical outcomes as some individuals respond excellently, others discreetly, or do not respond at all. Fifty consecutive COPD patients were enrolled. Of them, 35 starting a 5-week PR program were sampled at admission (T0), after 2 (T2W) and 5 (T5W) weeks, while 15 controls not yet on PR were tested at T0 and T5W. Nuclear magnetic resonance (NMR) profiling of exhaled breath condensate (EBC) and multivariate statistical analysis were applied to investigate the relationship between biomarkers and clinical parameters. The model including the three classes correctly located T2W between T0 and T5W, but 38.71% of samples partially overlapped with T0 and 32.26% with T5W, suggesting that for some patients PR is already beneficial at T2W (32.26% overlapping with T5W), while for others (38.71% overlapping with T0) more time is required. Rehabilitated patients presented several altered biomarkers. In particular, methanol from T0 to T5W decreased in parallel with dyspnea and fatigue, while the walk distance increased. Methanol could be ascribed to lung inflammation. We demonstrated that the metabolic COPD phenotype clearly evolves during PR, with a strict relationship between clinical and molecular parameters. Methanol, correlating with clinical parameters, represents a useful biomarker for monitoring personalized outcomes and establishing more targeted protocols

NMR Profiling of Exhaled Breath Condensate Defines Different Metabolic Phenotypes of Non-Cystic Fibrosis Bronchiectasis

Nuclear-magnetic-resonance (NMR) profiling of exhaled breath condensate (EBC) provides insights into the pathophysiology of bronchiectasis by identifying specific biomarkers. We evaluated whether NMR-based metabolomics discriminates the EBC-derived metabolic phenotypes ("metabotypes") of 41 patients with non-cystic fibrosis (nCF) bronchiectasis of various etiology [24 subjects with Primary Ciliary Dyskinesia (PCD); 17 patients with bronchiectasis not associated with PCD (nCF/nPCD)], who were compared to 17 healthy subjects (HS). NMR was used for EBC profiling, and Orthogonal Projections to Latent Structures with partial least-squares discriminant analysis (OPLS-DA) was used as a classifier. The results were validated by using the EBC from 17 PCD patients not included in the primary analysis. Different statistical models were built, which compared nCF/nPCD and HS, PCD and HS, all classes (nCF/nPCD-PCD-HS), and, finally, PCD and nCF/nPCD. In the PCD-nCF/nPCD model, four statistically significant metabolites were able to discriminate between the two groups, with only a minor reduction of the quality parameters. In particular, for nCF/nPCD, acetone/acetoin and methanol increased by 21% and 18%, respectively. In PCD patients, ethanol and lactate increased by 25% and 28%, respectively. They are all related to lung inflammation as methanol is found in the exhaled breath of lung cancer patients, acetone/acetoin produce toxic ROS that damage lung tissue in CF, and lactate is observed in acute inflammation. Interestingly, a high concentration of ethanol hampers cilia beating and can be associated with the genetic defect of PCD. Model validation with 17 PCD samples not included in the primary analysis correctly predicted all samples. Our results indicate that NMR of EBC discriminates nCF/nPCD and PCD bronchiectasis patients from HS, and patients with nCF/nPCD from those with PCD. The metabolites responsible for between-group separation identified specific metabotypes, which characterize bronchiectasis of a different etiology

Exploring Airway Diseases by NMR-Based Metabonomics: A Review of Application to Exhaled Breath Condensate

There is increasing evidence that biomarkers of exhaled gases or exhaled breath condensate (EBC) may help in detecting abnormalities in respiratory diseases mirroring increased, oxidative stress, airways inflammation and endothelial dysfunction. Beside the traditional techniques to investigate biomarker profiles, “omics” sciences have raised interest in the clinical field as potentially improving disease phenotyping. In particular, metabonomics appears to be an important tool to gain qualitative and quantitative information on low-molecular weight metabolites present in cells, tissues, and fluids. Here, we review the potential use of EBC as a suitable matrix for metabonomic studies using nuclear magnetic resonance (NMR) spectroscopy. By using this approach in airway diseases, it is now possible to separate specific EBC profiles, with implication in disease phenotyping and personalized therapy

An alien metabolite vs. a synthetic chemical hazard: an ecotoxicological comparison in the Mediterranean blue mussel

Bioactive natural products from marine invasive species may dramatically impact native communities, while many synthetic pharmaceutical drugs are released into the marine environment and have long-lasting harmful effects on aquatic life. Sometimes, metabolites from alien species and synthetic compounds share similar mechanisms of action, suggesting comparable ecotoxicological impacts. This applies to the alkaloid caulerpin (CAU) from the green alga Caulerpa cylindracea, highly invasive in the Mediterranean Sea, and to the synthetic lipid-lowering drug fenofibrate (FFB), both acting as agonists of peroxisome proliferator-activated receptors (PPARs). Analogies with FFB, which is widely considered hazardous to the aquatic environment, have led to concerns about the ecotoxicological potential of CAU. The problem has implications for public health as CAU is well known to enter the food web accumulating in fish of commercial importance. Here, we compared the effects of FFB and CAU through biochemical and histopathological analysis on a relevant bioindicator molluscan species, the mussel Mytilus galloprovincialis. Under laboratory conditions, mussels were fed with food enriched with CAU or FFB. After treatment, biochemical markers were analyzed revealing metabolic capacity impairments, cellular damage, and changes in acetylcholinesterase activity in mussels fed with FFB-enriched food. NMR-based metabolomic studies also showed significant alterations in the metabolic profiles of FFB-treated mussels. In addition, dietary administration of FFB produced morphological alterations in the mussels' gills and digestive tubules. Obtained results confirm that FFB is harmful to aquatic life and that its release into the environment should be avoided. Conversely, dietary treatment with CAU did not produce any significant alterations in the mussels. Overall, our results pave the way for the possible valorization of the huge biomass from one of the world's worst invasive species to obtain CAU, a natural product of interest in drug discovery.publishe

Modeling acquired resistance to the second-generation androgen receptor antagonist enzalutamide in the TRAMP model of prostate cancer

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this "TRAMP-based platform" to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC.Significance: Merging mathematical modeling with experimental data, this study presents the "TRAMP-based platform" as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases

Safety and Usage of C1-Inhibitor in Hereditary Angioedema: Berinert Registry Data

BackgroundThe plasma-derived, highly purified, nanofiltered C1-inhibitor concentrate (Berinert; “pnfC1-INH”) is approved in the United States for treating hereditary angioedema (HAE) attacks and in many European countries for attack treatment and short-term prophylaxis.ObjectiveThe objective of this study was to describe safety and usage patterns of pnfC1-INH.MethodsA multicenter, observational, registry was conducted between 2010 and 2014 at 30 United States and 7 European sites to obtain both prospective (occurring after enrollment) and retrospective (occurring before enrollment) safety and usage data on subjects receiving pnfC1-INH for any reason.ResultsOf 343 enrolled patients, 318 received 1 or more doses of pnfC1-INH for HAE attacks (11,848 infusions) or for prophylaxis (3142 infusions), comprising the safety population. Median dosages per infusion were 10.8 IU/kg (attack treatment) and 16.6 IU/kg (prophylaxis). Approximately 95% of infusions were administered outside of a health care setting. No adverse events (AEs) were reported in retrospective data. Among prospective data (n = 296 subjects; 9148 infusions), 252 AEs were reported in 85 (28.7%) subjects (rate of 0.03 events/infusion); 9 events were considered related to pnfC1-INH. Two thromboembolic events were reported in subjects with thrombotic risk factors. No patient was noted to have undergone viral testing for suspected blood-borne infection during registry participation.ConclusionsThe findings from this large, international patient registry documented widespread implementation of pnfC1-INH self-administration outside of a health care setting consistent with current HAE guidelines. These real-world data revealed pnfC1-INH usage for a variety of reasons in patients with HAE and showed a high level of safety regardless of administration setting or reason for use