Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation.

BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated. MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex® assay. RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation. DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

Sample optimization for saliva 1H-NMR metabolic profiling.

Nuclear Magnetic Resonance (NMR) based metabolomic analysis of whole saliva has provided potential diagnostic biomarkers for numerous human diseases contributing to a better understanding of their mechanisms. However, a comprehensive interpretation of the significance of metabolites in whole, parotid, and submandibular/sublingual saliva subtypes is still missing. Precision and reproducibility of sample preparation is an essential step. Here, we present a simple and efficient protocol for saliva 1H-NMR metabolic profiling. This procedure has been specifically designed and optimized for the identification and quantification of low concentration metabolites (as low as 1.1 µM) and is suitable for all the saliva subtypes.

Metabolic Profiles of Whole, Parotid and Submandibular/Sublingual Saliva.

The detection of salivary molecules associated with pathological and physiological alterations has encouraged the search of novel and non-invasive diagnostic biomarkers for oral health evaluation. While genomic, transcriptomic, and proteomic profiles of human saliva have been reported, its metabolic composition is a topic of research: metabolites in submandibular/sublingual saliva have never been analyzed systematically. In this study, samples of whole, parotid, and submandibular/sublingual saliva from 20 healthy donors, without dental or periodontal diseases, were examined by nuclear magnetic resonance. We identified metabolites which are differently distributed within the three saliva subtypes (54 in whole, 49 in parotid, and 36 in submandibular/sublingual saliva). Principal component analysis revealed a distinct cluster for whole saliva and a partial overlap for parotid and submandibular/sublingual metabolites. We found exclusive metabolites for each subtype: 2-hydroxy-3-methylvalerate, 3-methyl-glutarate, 3-phenylpropionate, 4-hydroxyphenylacetate, 4-hydroxyphenyllactate, galactose, and isocaproate in whole saliva; caprylate and glycolate in submandibular/sublingual saliva; arginine in parotid saliva. Salivary metabolites were classified into standard and non-proteinogenic amino acids and amines; simple carbohydrates; organic acids; bacterial-derived metabolites. The identification of a salivary gland-specific metabolic composition in healthy people provides the basis to invigorate the search for salivary biomarkers associated with oral and systemic diseases.

Red blood cells metabolome changes upon treatment with different X-ray irradiation doses.

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35-15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.

Statistical validation of 1H NMR protocol vs standard biochemical assay in quality control of RBC packed units.

BACKGROUND: Time dependent quantification of endogenous metabolites in biological samples (blood, urine, biological tissues extracts) in normal and pathological conditions as well as following therapeutic protocols is well established. In the clinical practice, such a dynamic flux of information allows the physician to identify and appreciate alterations associated to biochemical pathways of specific organs. In the years, many biochemical assays have been developed to detect, selectively, this vast array of molecules. METHODS: The Proton Nuclear Magnetic Resonance (1H NMR) spectrum allows the identification and quantification of more than 30 RBC-associated metabolites with minimum manipulation of the sample. To validate the use of 1H NMR spectroscopy for quality control purposes in transfusion medicine, a series of statistical tools have been employed to analyse and compare accuracy and precision of the 1H NMR results with respect to the ones obtained by standard biochemical assays. RESULTS: Among the many metabolites that can be detected and quantified by 1H NMR spectroscopy we selected creatinine and lactate, since they are routinely quantified by standard biochemical assays and because they are characterized by a wide concentration dynamic range. We show that 1D 1H NMR spectroscopy is an accurate a precise method for metabolite quantification. CONCLUSION: These results validate the use of 1H NMR spectroscopy in transfusion medicine as a method to evaluate the quality of RBC packed units and to develop novel and more efficient RBCs storage protocols.

The allergen Mus m 1.0102: Dissecting the relationship between molecular conformation and allergenic potency.

BACKGROUND: The species Mus musculus experiences an obligate proteinuria: predominant are the Major Urinary Proteins (MUPs), that, collectively known as the major mouse allergen Mus m 1, are among the most important aeroallergens for mouse allergic patients. The production of a soluble and stable hypoallergenic form of Mus m 1 is essential for the development of immunotherapeutic protocols to treat allergic symptoms. METHODS: We introduced the substitution C138S in recombinant Mus m 1.0102, an allergenic isoform of Mus m 1. Solubility, conformation, stability and ability to refold after chemical denaturation were investigated with dynamic light scattering, circular dichroism, fluorescence and NMR spectroscopy. An in vitro degranulation assay was used to evaluate the protein allergenic potential, and compare it with Mus m 1.0102 and with an hypoallergenic variant bearing the substitution Y120A. RESULTS: Mus m 1.0102-C138S retains a native-like fold revealing, however, local conformational alterations that influence some of its physical and allergenic properties: it is monodispersed, thermostable up to 56°C, able to reversibly unfold and it exhibits an enhanced allergenicity. CONCLUSIONS: The unique free thiol group affects the solution structural stability of the native protein. Because the mutant C138S does not aggregate over time it is a good lead protein to develop diagnostic and therapeutic applications. GENERAL SIGNIFICANCE: We elucidated the relationship between unfolding reversibility and sulphydryl reactivity. We ascribed the enhanced allergenicity of the mutant C138S to an increased accessibility of its allergenic determinants, an enticing feature to further investigate the structural elements of the allergen-IgE interface.

A Comparative Study of the Effect of Leukoreduction and Pre-storage Leukodepletion on Red Blood Cells during Storage.

Blood transfusion is a fundamental therapy in numerous pathological conditions. Regrettably, many clinical reports describe adverse transfusion's drawbacks due to red blood cells alterations during storage. Thus, the possibility for a blood bank to ameliorate the quality of the erythrocyte concentrates units is crucial to improve clinical results and reduce transfusion adverse occurrences. Leukodepletion is a pre-storage treatment recognized to better preserve the quality of red blood cells with respect to leukoreduction. Aim of this work is to unravel the biochemical and biophysical basis that sustain the good clinical outcomes associated to the use of leukodepleted erythrocytes units. Erythrocytes concentrates were prepared as leukoreduced (n = 8) and pre-storage leukodepleted (n = 8) and then studied during 6 weeks in blood bank conditions. Overall, the data indicate that leukodepletion not only provide red blood cells with an appropriate amount of nutrients for a longer time but also selects red blood cells characterized by a more resilient plasma membrane fit to prolong their viability. We believe these results will stimulate new ideas to further optimize the current storage protocols.

Biochemical assessment of red blood cells during storage by (1)H nuclear magnetic resonance spectroscopy. Identification of a biomarker of their level of protection against oxidative stress.

BACKGROUND: Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions. MATERIALS AND METHODS: Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of (1)H-NMR spectra. RESULTS: The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile. DISCUSSION: We proved the analytical power of (1)H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.

In search of a vaccine for mouse allergy: significant reduction of Mus m 1 allergenicity by structure-guided single-point mutations.

BACKGROUND: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. METHODS: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. RESULTS: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. CONCLUSIONS: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes.

(15)N and (1)HN chemical shift data and (15)N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the beta-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the beta-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.