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The identification of markers of inflammatory activity at the early stages of pulmonary diseases which share common characteristics that prevent their clear differentiation is of great significance to avoid misdiagnosis, and to understand the intrinsic molecular mechanism of the disorder. The combination of electrophoretic/chromatographic methods with mass spectrometry is currently a promising approach for the identification of candidate biomarkers of a disease. Since the fluid phase of sputum is a rich source of proteins which could provide an early diagnosis of specific lung disorders, it is frequently used in these studies. This report focuses on the state-of-the-art of the application, over the last ten years (2011–2021), of sputum proteomics in the investigation of severe lung disorders such as COPD; asthma; cystic fibrosis; lung cancer and those caused by COVID-19 infection. Analysis of the complete set of proteins found in sputum of patients affected by these disorders has allowed the identification of proteins whose levels change in response to the organism’s condition. Understanding proteome dynamism may help in associating these proteins with alterations in the physiology or progression of diseases investigated.

Background Vascular Ehlers–Danlos syndrome (vEDS) is a heritable connective tissue disorder caused by defects in the type III collagen protein. It is generally considered the most severe form of Ehlers–Danlos syndrome (EDS) due to an increased risk of spontaneous artery or organ rupture. vEDS has an extremely heterogeneous presentation and muscle rupture is considered a minor diagnostic criterium. Methods A patient with a long history of inconclusive examinations and investigations was referred to our unit. The clinical picture was mainly characterized by muscle ruptures, whereas the cardiovascular involvement was limited to mitral regurgitation. We performed a panel analysis of genes associated with inheritable heart diseases using the TruSight Cardio kit (Illumina). A skin biopsy was then performed for functional studies to analyze the different forms of collagen molecules produced in vitro by cutaneous fibroblasts. Results The patient presented the novel variant c.3478A>G (p.Ile1160Val) in COL3A1 (NM_000090.3), whose pathogenicity was supported by biochemical analysis of type III collagen. Conclusion In this report, we describe a case of vEDS with predominant and severe musculoskeletal involvement. Our findings provide insight into genetic variants and clinical expression of vEDS, broadening the clinical scenario of the syndrome., In this report, we describe a case of vEDS with predominant and severe musculoskeletal involvement. Our findings provide insight into genetic variants and clinical expression of vEDS, enlarging the clinical scenario of the syndrome.

Detection of proteins which may be potential biomarkers of disorders represents a big step forward in understanding the molecular mechanisms that underlie pathological processes. In this context proteomics plays the important role of opening a path for the identification of molecular signatures that can potentially assist in early diagnosis of several clinical disturbances. Aim of this report is to provide an overview of the wide variety of proteomic strategies that have been applied to the investigation of chronic obstructive pulmonary disease (COPD), a severe disorder that causes an irreversible damage to the lungs and for which there is no cure yet. The results in this area published over the past decade show that proteomics indeed has the ability of monitoring alterations in expression profiles of proteins from fluids/tissues of patients affected by COPD and healthy controls. However, these data also suggest that proteomics, while being an attractive tool for the identification of novel pathological mediators of COPD, remains a technique mainly generated and developed in research laboratories. Great efforts dedicated to the validation of these biological signatures will result in the proof of their clinical utility.

Background and Aims Adenosine (Ado), the main substrate of ATP, is a potent endogenous anti-inflammatory nucleoside. Hypoxia induces ATP depletion, AMP extracellular increase that is phosphohydrolyzed to ADO by the enzyme ecto-5′-nucleotidase (CD73). Constitutive CD73 expression is higher in deep cortex outer medulla that is the most vulnerable region to ischemic injury. Notably CD73 is also expressed on Mesenchymal Stromal Cell (MSC) and is essential phenotypic marker. Previously in a rat model of Donation after Circulatory Death (DCD), we demonstrated that the perfusion of ischemic kidney with MSC or MSC derived Extracellular Vesicles (EV) protects tissue up regulating mitochondrial energetic metabolism genes. The aim of this study was to investigate the role of CD73/Ado system in MSC/EV protection from ischemia. Method Fisher rats were used as kidney donors and Lewis rats as MSC donors. MSC missing CD73 were produced by electroporation in the presence of specific siRNA to block CD73 expression (siMSC). EV were isolated from supernatants of MSC and siMSC (siEV) medium through differential ultracentrifugations. Size distribution and enumeration analysis were performed using NanoSight NS300. EV phenotypic characterization was performed in flow cytometer using CD45, CD49e, CD63, CD9, CD81 and CD73 monoclonal antibodies. DCD model was obtained by renal artery clamping for 20 minutes. This warm ischemia time represents the “no touch period” imposed by the Italian law to death declaration. After nephrectomy, kidneys were perfused in hypothermia (4°C) with Belzer Solution (BS), or BS supplemented with 3x106 MSC (MSC) or BS supplemented with 28,5x109 EV/siEV (EV/siEV). The effluent fluid (EF) was collected at the beginning (T0), every hours (T1, T2, T3) and at the end of the perfusion (T4). Ado and ATP determinations in EF and tissues were performed by HPLC and ELISA, respectively. Results EF Ado was significantly higher in MSC vs BS from T1 and in EV vs BS from T0 to T4 (p Conclusion CD73 expressed on MSC /EV impacts on cell energy metabolism pathway and ATP generation. This is the first evidence that MSC/EV act through CD73/Ado system to prevent ischaemic damage.

BACKGROUND: Postmenopausal women experience undesired symptoms that adversely affect their quality of life. In the recent years, a specific 12 - week fractional CO2 laser treatment has been introduced, with highly significant relief of symptoms.AIM: The aim of this paper is the identification of the early modifications of structural components of atrophic vaginal mucosa induced by laser irradiation, which is responsible for the restorative processes.MATERIAL AND METHODS: We investigated by microscopical, ultrastructural and biochemical methods the modifications of the structural components of postmenopausal atrophic vaginal mucosa tissues after 1 hour following a single fractional laser CO2 application.RESULTS: In one hour, the mucosal epithelium thickens, with the maturation of epithelial cells and desquamation at the epithelial surface. In the connective tissue, new papillae indenting the epithelium with newly formed vessels penetrating them, new thin fibrils of collagen III are also formed in a renewed turnover of components due to the increase of metalloproteinase - 2. Specific features of fibroblasts support stimulation of their activity responsible of the renewal of the extracellular matrix, with an increase of mechanical support as connective tissue and stimulation of growth and maturation to epithelium thanks to new vessels and related factors delivered.CONCLUSION: We found the activation of regenerative mechanisms expressed both in the connective tissue - with the formation of new vessels, new papillae, and new collagen - and in the epithelium with the associated thickening and desquamation of cells at the mucosal surface.

Nasu-Hakola Disease (NHD) is a recessively inherited systemic leukodystrophy disorder characterized by a combination of frontotemporal presenile dementia and lytic bone lesions. NHD is known to be genetically related to a structural defect of TREM2 and DAP12, two genes that encode for different subunits of the membrane receptor signaling complex expressed by microglia and osteoclast cells. Because of its rarity, molecular or proteomic studies on this disorder are absent or scarce, only case reports based on neuropsychological and genetic tests being reported. In light of this, the aim of this paper is to provide evidence on the potential of a label-free proteomic platform based on the Multidimensional Protein Identification Technology (MudPIT), combined with in-house software and on-line bioinformatics tools, to characterize the protein expression trends and the most involved pathways in NHD. The application of this approach on the Lymphoblastoid cells from a family composed of individuals affected by NHD, healthy carriers and control subjects allowed for the identification of about 3000 distinct proteins within the three analyzed groups, among which proteins anomalous to each category were identified. Of note, several differentially expressed proteins were associated with neurodegenerative processes. Moreover, the protein networks highlighted some molecular pathways that may be involved in the onset or progression of this rare frontotemporal disorder. Therefore, this fully automated MudPIT platform which allowed, for the first time, the generation of the whole protein profile of Lymphoblastoid cells from Nasu-Hakola subjects, could be a valid approach for the investigation of similar neurodegenerative diseases.

Purpose Bronchiolitis obliterans Syndrome (BOS) is a major long term complication after lung transplant. The role of Neutrophil (Neu) activation in BOS pathogenesis has been repeatedly suggested but needs to be fully clarified. Human Neutrophil Elastase (HNE) as well as complexes of alpha-1-antitrypsin (AAT)/HNE are more frequently detected in BAL-f from BOS patients thus suggesting chronic Neu activation BOS. NETosis is a mechanism of Neu response to pathogens which can drive severe epithelial injury. Here, we aim to analyze possible role of NETosis in BOS by assessing number of Neu and related markers in BAL-f. Methods A total of 16 BAL samples from BOS (n° 8, 3 BOS1, 2 BOS2 and 2 BOS3) and from stable lung recipients (n°8) were included. Patients with sarcoidosis (Sarco) were used as control (n°3). The following measures were performed: BAL Neu counts; HNE, IL1beta and IL8 (ELISA); NETs (measuring HNE-DNA complexes by ELISA); AAT/HNE complexes (WB). Results NETs were measurable in all BAL samples from Tx recipients, with a not-significant trend towards higher values among BOS patients (p=0.275). Interestingly NETs levels did not correlate with Neu counts (r=0.575; p=0.143) but, limited to BOS patients, significantly correlated with IL-8 (r=0.786; p=0.028), HNE (r=0.736; p=0.028), and AAT/HNE complexes (r=0.73; p=0.05).No correlation with IL1beta levels was found. Conclusion This is the first preliminary evidence of the possibility to detect NETs in BAL-f of lung transplant recipients and, interestingly points out that NETs levels do not seem to correlate simply with Neu counts but rather with inflammation and specific Neu activation. Thus, they could potentially represent a marker of higher inflammatory state. In addition, given the evidence that NETs drive epithelial cell injury and cause EMT their significant pathogenic role in BOS can be suggested and future therapeutic strategies can be hypothesized.

Aim of this article is to focus the attention of the reader on the application of CE/MS and LC/MS to the analysis of human body fluids not currently used for the diagnosis of disorders and, for this reason, catalogued as "less/nonconventional" fluids, that is, tears, nasal secretions, cerumen, bronchoalveolar lavage fluid, sputum, exhaled breath condensate, nipple aspirate, breast milk, amniotic fluid, bile, seminal plasma, liposuction aspirate fluid, and synovial fluid. The pool of articles presented in this report demonstrates that, rather than being neglected, these fluids are an important resource for the evaluation of possible pathologic conditions. Thus, being a sort of mirror that reflects the normal internal characteristics and disease state of an individual, they benefit of an increasing appreciation. This review follows a previous report of this series and covers the latest developments in this field that have been published in specialist journals in the years 2015-2017.

This report reviews the literature of the past decade dealing with the combination of electrokinetic and chromatographic strategies in the proteomic field. Aim of this article is to highlight how the application of complementary techniques may contribute to substantially improve protein identification. Several studies here considered demonstrate that exploring the combination of these approaches can be a strategy to enrich the extent of proteomic information achieved from a sample. The coupling of "top-down" and "bottom-up" proteomics may result in the generation of a hybrid analytical tool, very efficient not only for large-scale profiling of complex proteomes but also for studying specific subproteomes. The range of applications described, while evidencing a continuous boost in the imagination of researchers for developing new combinations of methods for protein separation, also underlines the adaptability of these techniques to a wide variety of samples. This report points out the general usefulness of combining different procedures for proteomic analysis, an approach that allows researchers to go deeper in the proteome of samples under investigation.

Colour polymorphism occurs when two or more genetically-based colour morphs permanently coexist within an interbreeding population. Colouration is usually associated to other life-history traits (ecological, physiological, behavioural, reproductive …) of the bearer, thus being the phenotypic marker of such set of genetic features. This visual badge may be used to inform conspecifics and to drive those decision making processes which may contribute maintaining colour polymorphism under sexual selection context. The importance of such information suggests that other communication modalities should be recruited to ensure its transfer in case visual cues were insufficient. Here, for the first time, we investigated the potential role of proteins from femoral gland secretions in signalling colour morph in a polymorphic lizard. As proteins are thought to convey identity-related information, they represent the ideal cues to build up the chemical modality used to badge colour morphs. We found strong evidence for the occurrence of morph-specific protein profiles in the three main colour-morphs of the common wall lizard, which showed both qualitative and quantitative differences in protein expression. As lizards are able to detect proteins by tongue-flicking and vomeronasal organ, this result support the hypothesis that colour polymorphic lizards may use a multimodal signal to inform about colour-morph.

The present review intends to cover the literature on the use of CE-/LC-MS for the analysis of human fluids, from 2010 until present. It has been planned to provide an overview of the most recent practical applications of these techniques to less extensively used human body fluids, including, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate, tear fluid, breast fluid, amniotic fluid, and cerumen. Potential pitfalls related to fluid collection and sample preparation, with particular attention to sample clean-up procedures, and methods of analysis, from the research laboratory to a clinical setting will also be addressed. While being apparent that proteomics/metabolomics represent the most prominent approaches for global identification/quantification of putative biomarkers for a variety of human diseases, evidence is also provided of the suitability of these sophisticated techniques for the detection of heterogeneous components carried by these fluids.

BACKGROUND: Postmenopausal women experience undesired symptoms that adversely affect their quality of life. In the recent years, a specific 12 - week fractional CO2 laser treatment has been introduced, with highly significant relief of symptoms. AIM: The aim of this paper is the identification of the early modifications of structural components of atrophic vaginal mucosa induced by laser irradiation, which is responsible for the restorative processes. MATERIAL AND METHODS: We investigated by microscopical, ultrastructural and biochemical methods the modifications of the structural components of postmenopausal atrophic vaginal mucosa tissues after 1 hour following a single fractional laser CO2 application. RESULTS: In one hour, the mucosal epithelium thickens, with the maturation of epithelial cells and desquamation at the epithelial surface. In the connective tissue, new papillae indenting the epithelium with newly formed vessels penetrating them, new thin fibrils of collagen III are also formed in a renewed turnover of components due to the increase of metalloproteinase - 2. Specific features of fibroblasts support stimulation of their activity responsible of the renewal of the extracellular matrix, with an increase of mechanical support as connective tissue and stimulation of growth and maturation to epithelium thanks to new vessels and related factors delivered. CONCLUSION: We found the activation of regenerative mechanisms expressed both in the connective tissue - with the formation of new vessels, new papillae, and new collagen - and in the epithelium with the associated thickening and desquamation of cells at the mucosal surface.

Muscle release of the amino acid 3-methyl-histidine (3MH) is a sensitive index of myofibrillar protein overdegradation (MPO). We hypothesized that patients with chronic heart failure (CHF) could have increased muscle release of 3MH, which in turn reflects MPO, and that serum electrolyte sodium (Na(+)) and potassium (K(+)) levels may be associated with this 3MH muscle release.Thirty-one overweight outpatients (body mass index, 27 ± 4.4 kg/m(2); 22 men and 9 women; age, 56 ± 8.7 y) with clinically stable CHF were studied. After a 24-hour meat-free diet and overnight fasting, patients underwent blood sampling from a cannulated arm vein (V) and concomitantly from the arterial artery (A) to determine plasma 3MH levels and to calculate the A-V difference. Serum levels of Na(+) and K(+) in the venous blood were determined, and the Na(+)/K(+) ratio was calculated. Ten healthy subjects who were matched for gender, age, and body mass index served as controls and underwent the same protocol as the patients with CHF.The patient group had higher arterial (P = 0.02) and venous (P = 0.005) 3MH levels but a similar A-V 3MH difference (P = 0.28) as compared with the controls. Within the CHF group, 67.7% of patients released 3MH, which resulted in a negative A-V value (P0.02 as compared with controls). In patients with CHF, the A-V 3MH difference correlated positively with the serum K(+) level (r = 0.62; P = 0.0002) and negatively with Na(+)/K(+) ratio (r = -0.55; P = 0.002). No association was found between the A-V 3MH difference and the Na(+) level.The study demonstrated the existence of MPO in resting overweight patients with CHF, thereby suggesting that low serum levels of K(+) may contribute to MPO.

To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004–2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.

This report reviews the literature of the past decade dealing with the combination of electrokinetic and chromatographic strategies in the proteomic field. Aim of this article is to highlight how the application of complementary techniques may contribute to substantially improve protein identification. Several studies here considered demonstrate that exploring the combination of these approaches can be a strategy to enrich the extent of proteomic information achieved from a sample. The coupling of "top-down" and "bottom-up" proteomics may result in the generation of a hybrid analytical tool, very efficient not only for large-scale profiling of complex proteomes but also for studying specific subproteomes. The range of applications described, while evidencing a continuous boost in the imagination of researchers for developing new combinations of methods for protein separation, also underlines the adaptability of these techniques to a wide variety of samples. This report points out the general usefulness of combining different procedures for proteomic analysis, an approach that allows researchers to go deeper in the proteome of samples under investigation.

The neutrophilic component in bronchiolitis obliterans syndrome (BOS, the main form of chronic lung rejection), plays a crucial role in the pathogenesis and maintenance of the disorder. Human Neutrophil Elastase (HNE), a serine protease responsible of elastin degradation whose action is counteracted by &alpha, 1-antitrypsin (AAT), a serum inhibitor specific for this protease. This work aimed to investigate the relationship between HNE and AAT in bronchoalveolar lavage fluid (BALf) from stable lung transplant recipients and BOS patients to understand whether the imbalance between proteases and inhibitors is relevant to the development of BOS. To reach this goal a multidisciplinary procedure was applied which included: (i) the use of electrophoresis/western blotting coupled with liquid chromatography-mass spectrometric analysis, (ii) the functional evaluation of the residual antiprotease activity, and (iii) a neutrophil count.

Desmosine (DES) and isodesmosine are two isomer amino acids unique-to-mature, cross-linked elastin. Based on this feature, they have been discussed as surrogate markers of chronic obstructive pulmonary disease, a disorder characterized by progressive degradation of lung elastin. Despite the development of numerous protocols, detection of DESs in body fluids is still considered to be technically challenging. In fact, owing to the minute concentration of these circulating cross-links, their accurate measurement may be provided only by sophisticated and sensitive techniques. Aim of this article is to present the "history" of the two techniques (MEKC and LC-MS) that, better than others, allowed scientists to "bring their best to the table" in this area. Both of them meet the criteria of (almost) complete automation of the procedure and of the use of more selective and sensitive detection systems. The substantial advantages in terms of precision and accuracy provided by such measurements suggest that the science of DESs is eventually catching up with its promise and the assumption that these candidate biomarkers can be associated to clinical variables holds true.

The purpose of this study was to investigate whether the documented difficulties of physiological amounts of essential amino acids (EAAs) (7 g) to induce protein synthesis could be reflected in a simple method adaptable to a clinical setting. Sixteen healthy individuals, nine elderly (75.3 ± 3.5 years), and seven young (28 ± 2.5 years) were enrolled in the study. Five minutes before EAA ingestion (baseline) and 20, 40, 60, 90, 120, 180 min after EAA ingestion, venous blood samples were taken from the ante-cubital vein to determine the concentrations of EAAs (μmol/L). The results show that plasma EAA increases were significantly higher in old than in young persons at the considered time points (from p

Three pools of exhaled breath condensate (EBC) from non-smokers plus healthy smokers (NS + HS, n = 45); chronic obstructive pulmonary disease (COPD) without emphysema (COPD, n = 15) and subjects with pulmonary emphysema associated with α(1)-antitrypsin deficiency (AATD, n = 23) were used for an exploratory proteomic study aimed at generating fingerprints of these groups that can be used in future pathophysiological and perhaps even clinical research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the platform applied for this hypothesis-free investigation. Analysis of pooled specimens resulted in the production of a "fingerprint" made of 44 proteins for NS/HS; 17 for COPD and 15 for the group of AATD subjects. Several inflammatory cytokines (IL-1α, IL-1β, IL-2; IL-12, α and β subunits, IL-15, interferon α and γ, tumor necrosis factor α); Type I and II cytokeratins; two SP-A isoforms; Calgranulin A and B and α1-antitrypsin were detected and validated through the use of surface enhanced laser-desorption ionization mass spectrometry (SELDI-MS) and/or by Western blot (WB) analysis. These results are the prelude of quantitative studies aimed at identifying which of these proteins hold promise as identifiers of differences that could distinguish healthy subjects from patients.

The proteins in the lungs are in constant flux, undergoing degradation and resynthesis. We investigated pulmonary protein and amino acid metabolism, the biochemical basis of the remodeling process, in individuals with chronic heart failure receiving or not receiving β-blocker therapy with bisoprolol (BIS).Clinically stable rehabilitative patients with chronic heart failure, without metabolic diseases or liver/renal failure, and with a stable weight over the preceding 3 mo underwent right heart catheterization, and radial artery cannulation. Mixed central venous and arterial blood samples were drawn simultaneously to calculate the venous-arterial difference of amino acids (pulmonary uptake and release).Twenty-two patients on BIS therapy and eight not receiving BIS were analyzed. The two groups showed a net pulmonary protein synthesis (i.e., a positive value of phenylalanine [venous-arterial difference] × cardiac index product) and amino acid extraction, the rates of which were significantly lower in patients on BIS therapy. The two groups had pulmonary hypertension (mean pulmonary artery pressure19 mmHg). Pulmonary vascular resistance was 57% higher in patients not receiving BIS than in those on BIS therapy (6.65 ± 2.90 versus 4.23 ± 1.49 mmHg/L · min⁻¹ · m⁻², P0.05). Pulmonary vascular resistance correlated positively with the pulmonary extraction of total essential amino acids (r = +0.4576, P = 0.01) and leucine (r = +0.5083, P = 0.004), the most important amino acid for protein synthesis.Patients with chronic heart failure have increased rates of amino acid extraction and pulmonary protein synthesis, suggesting, at least in part, an increased rate of lung remodeling. Therapy with BIS attenuates lung metabolic abnormalities.

There is a pressing need to identify biomarkers that are useful to monitor effects of expensive treatments such as i.v. alpha-1-antitrypsin (AAT) augmentation. We applied Nuclear Magnetic Resonance (NMR) spectroscopy to single , blinded samples of exhaled breath condensate (EBC) from 11 patients with moderate to severe emphysema and PiZZ- AAT deficiency and 11 spouse healthy controls. The spectral metabolomics profiles showed the presence of 20 to 25 metabolites such as propionic acid, acetate, butyrate, benzoate, butanediol, fatty acid, alanine, acetone, acetoin, ethanol, lactate and methanol which were common to both groups. However, the difference in the level of metabolites (4 to 300 fold, p PLS-DA analysis: 2D scores plot between the selected PCs.

Purpose: Tools that will help unraveling the complexity of Bronchiolitis Obliterans Syndrome (BOS) and identifying useful predictive markers are urgently needed. Given its ability of capturing disease-relevant metabolic profile changes, Metabolomics may occupy an important role in this area. Methods: BALf was obtained fromlung transplant patients, before and after BOS development,and from subjects affected by other respiratory diseases. Samples were submitted to NMR analyses,through the acquisition of mono and bi-dimensional spectra allowing the observation of 1 H and 13 C nuclei. Results: Analyses were performed on both BAL and BALf from patients with different respiratory conditions (58 BOS, 4 sarcoidosis, 4 AAE, 2 pulmonary nodules). Exploiting the combination of mono and 2-Dexperiments, NMRspectra allowed the unequivocal identification and quantification of 36 polar metabolites,includingaminoacids, monosaccharides, nucleotides, Krebs cycle intermediates and phospholipid precursors. The resonances of about 10 additional metabolites have not been assigned yet. Fluctuations in the concentration of specific metabolites were observed not only among subjects with different pathologies but also within the cohort of BOS patients. Conclusions: These preliminary results,while underlining the potential of NMR to identify biomarkers of lung pathologies,strongly support the conviction that this analysis onBALf from BOS patients may provide metabolic signatures of the disease. We demonstrate thatNMR is also suitable to perform metabolic profiling ofBALincluding cells and fluid components. Acknowledgements: supported by a CARIPLO (Milan, Italy) grant#2013-0820, Bando 2013-Ricerca Medica.

To investigate whether supplementation with oral essential amino acids (EAAs) may reduce the occurrence of nosocomial infection among patients with brain injury (BI: stroke, trauma, anoxic coma).Patients (n = 125; 77 men, 48 women; mean age 63 ± 15 years) with stroke (68.8%), subarachnoid hemorrhage (17.6%), traumatic BI (7.2%), and anoxic BI (6.4%) 88 ± 15 days after the index event. Patients were randomly assigned to 2 months of oral EAAs (n = 63; 8 g/d) or placebo (n = 62).Over the first month of rehabilitation, there were 60 infections in the whole population of 125 patients (48%); however, the rate was 23.2% lower in the EAA group (23 episodes/63 patients; 36.5%) than in the placebo group (37 episodes/62 patients; 59.7%) (P.01). The types of infection were similarly distributed between the 2 groups. Serum levels of prealbumin20 mg/dL and C-reactive protein (CRP)0.3 mg/dL were the best predictors of future infection (prealbumin: odds ratio [OR] = 4.17, confidence interval [CI] 1.84-9.45, P.001; CRP: OR = 3.8, CI 1.71-8.44, P.001).Supplementary EAAs may reduce the occurrence of nosocomial infections in rehabilitation patients with BI. Prealbumin and CRP are the best predictors of future infections.

The very high general infection rate (IRI) observed in our Geriatric Intensive Rehabilitation Center (GIRC) led us to investigate whether patient supplementation with essential amino acids (EAAs), modulators of immuno-competence, could reduce IRI. Eighty elderly patients admitted to our GIRC (n=40; age 79.5 ± 7.71; male/female 14/26) or placebo (n=40; age 82.13 ± 6.15; male/female 13/27) were allocated to an 8 g/day oral EAAs group and were surveyed for infections (48 h from admission) over the first month of their hospital stay. The IRI was 67% for the entire population of patients, 82.5% (33/40 patients) in the placebo group and 52% (21/40 patients) in the EAA group (p0.02). When patients were divided into infection group (IG) and without-infection group (WIG), independently of post randomization allocation, the WIG had higher levels of serum albumin (p0.001), blood hemoglobin (Hb) concentration (p=0.01), dietary protein (p=0.008) calorie intakes (p=0.05) but lower serum C-reactive protein (CRP) (p0.001). The factor of CRP0.8 mg/dl and Hb ≤ 12 in females, ≤13 in males was associated 4 times and 3.6 times risk of infection, respectively, by sex. EAAs supplementation may lower the risk of infection by 30% in the rehabilitative elderly population. CRP and blood hemoglobin levels can be considered risk markers of future infection.

BackgroundIn infected lungs of the cystic fibrosis (CF) patients, opportunistic pathogens and mutated cystic fibrosis transmembrane conductance regulator protein (CFTR) contribute to chronic airway inflammation that is characterized by neutrophil/macrophage infiltration, cytokine release and ceramide accumulation. We sought to investigate CF lung inflammation in the alveoli.MethodsLung tissue from 14 CF patients and four healthy individuals was analyzed for numbers of effector cells, elastin and collagen concentrations, inflammatory markers and density of Pseudomonas aeruginosa. Additionally, desmosine and isodesmosine concentrations were determined in 52 urine specimens from CF patients to estimate the burden of elastase activities in respiratory secretions.ResultsElastin concentration was significantly decreased and collagen significantly increased in CF alveolar tissues as compared to age-matched, healthy individuals. Elastin split products were significantly increased in urine samples from patients with CF and correlated inversely with age, indicating local tissue remodelling due to elastin degradation by unopposed proteolytic enzymes. Alveolar inflammation was also characterized by a significant cell infiltration of neutrophils, macrophages and T cells, extensive nuclear factor-κB and insulin-like growth factor-1 activation in various cell types and increased intercellular adhesion molecule-1 expression, and increased numbers of myofibroblasts. Additionally, ceramide accumulated in type II alveolar epithelial cells, lacking CFTR. P. aeruginosa organisms were rarely present in inflamed alveoli.ConclusionsChronic inflammation and remodeling is present in alveolar tissues of the CF lung and needs to be addressed by anti-inflammatory therapies.

Background: An adequate energy-protein intake (EPI) when combined with amino acid supplementation may have a positive impact nutritional and metabolic status in patients with chronic heart failure (CHF). Methods and results: Thirty eight stable CHF patients (27 males, 73.5±4 years; BMI 22.5±1.4 kg/m2), with severe depletion of muscle mass and were randomised to oral supplements of essential amino acids 8 g/day (EAA group; n=21) or no supplements (controls; n=17). patients had adequate EPI (energy ≥ 30 kcal/kg; proteins >1.1 g/kg). At baseline and 2-months after randomisation, the patients underwent metabolic (plasma lactate, pyruvate concentration; serum insulin level; estimate of insulin resistance by HOMA index), nutritional (measure of nitrogen balance), and functional (exercise test, walking test) evaluations. Body weight increased by >1 kg in 80% of supplemented patients (mean 2.96 kg) and in 30% of controls (mean 2.3 kg) (interaction

Introduction: The objective of this study was to investigate whether protein-calorie supplementation may enhance the cognitive retrieval of patients with stroke. Patients and methods: A randomized, double-blind, controlled pilot clinical trial was performed comparing diet and diet plus protein-calorie supplementation regimens. The subjects were 48 patients with subacute stroke (≥ 14 days from index event). Anthropometric and nutritional (3-day diary) variables, cognitive function (Mini-Mental State Examination; MMSE) were determined before and 21 days after randomization in control and daily supplemented group (formula providing 250 kcal + 20 g protein). Results: At day 21 after starting the protocol, only the supplemented group significantly improved their performance to MMSE (log10MMSE +0.6 ± 0.4 score; P = 0.01 from baseline). Conclusions: Protein-calorie supplementation may enhance the recovery of cognitive function in subacute stroke patients.

Desmosines are crosslinking amino acids unique to mature elastin in humans. Owing to this unicity, they have been discussed as potentially attractive indicators of connective tissue disorders whose clinical manifestations are mostly the result of elastin degradation. This review covers advances in immunochemical, chromatographic, and electrophoretic procedures applied in the last 25 years to detect and quantitate these crosslinksin a variety of biological samples. Recent applications of CE with LIF detection (CE-LIF) for investigating the content of desmosines in different fluids will also be discussed.

We aimed to document in stroke patients peripheral blood immune cell profiles, their relations with neuro-functional tests, and any possible influence of supplemented essential amino acids (EAAs) may have on both the immune system and the relationship of the latter with neuro-function. Forty-two dysphagic stroke patients (27 men; 71±9 years) underwent bio-humoral measurements, neuro-functional tests, including Functional Independence Measure (FIM) and Dysphagia Outcome and Severity Scale (DOSS), and were randomized to receive EAAs 8 g/d (EAA group) or isocaloric maltodextrin (placebo group). At discharge all measurements were repeated 38±1 days after randomization. At admission, total white cell (TWC), neutrophil (N), and lymphocyte (Lymph) counts were normal and the N/Lymph ratio was higher than normal values ( For the regression analysis, the overtime changes of Lymph % TWC correlated significantly with DOSS ( P = 0.01). There was a positive correlation between Lymph % TWC and DOSS for the entire stroke population ( P = 0.015). While this correlation was not important for the placebo group ( P = 0.27), it was significant in the EAA subgroup ( P = 0.018). In the sub-acute stroke stage, there may be slight alterations of peripheral blood immune cells. Lymph cells are associated with improved neuro-function tests with evidence that this association is enhanced by supplementing EAAs.

The present review intends to cover the literature on the use of CE-/LC-MS for the analysis of human fluids, from 2010 until present. It has been planned to provide an overview of the most recent practical applications of these techniques to less extensively used human body fluids, including, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate, tear fluid, breast fluid, amniotic fluid, and cerumen. Potential pitfalls related to fluid collection and sample preparation, with particular attention to sample clean-up procedures, and methods of analysis, from the research laboratory to a clinical setting will also be addressed. While being apparent that proteomics/metabolomics represent the most prominent approaches for global identification/quantification of putative biomarkers for a variety of human diseases, evidence is also provided of the suitability of these sophisticated techniques for the detection of heterogeneous components carried by these fluids.

Mutations in COL1A1 and COL1A2 genes, encoding the alpha1 and alpha2 chain of type I collagen, respectively, are responsible for the vast majority of cases of osteogenesis imperfecta (OI) (95% of patients with a definite clinical diagnosis). We have investigated 22 OI patients, representing a heterogeneous phenotypic range, at the biochemical and molecular level. A causal mutation in either type I collagen gene was identified in 20 of them: no recurrent mutation was found in unrelated subjects; 15 out of 20 mutations had not been reported previously. In two patients, we could not find any causative mutation in either type I collagen gene, after extensive genomic DNA sequencing. Failure of COL1A1/COL1A2 mutation screening may be due, in a few cases, to further clinical heterogeneity, i.e. additional non-collagenous disease loci are presumably involved in OI types beyond the traditional Sillence's classification.

The aim of the present article is to review the efforts performed in the past two decades by numerous research groups for the development of methods that allow a correct diagnosis of prolidase deficiency (PD), a rare autosomal recessive disorder and for the rationalization of a possible therapeutic intervention on these patients. In particular, the interest of the reader is focused on the application of capillary electrophoresis (i) for the detection of biological markers that reflect the pathological feature of the disease and (ii) for the determination of the efficiency of a carrier system in delivering prolidase inside cells in a possible therapy based on enzyme replacement.

Human urine plays a central role in clinical diagnostic being one of the most-frequently used body fluid for detection of biological markers. Samples from patients with different diseases display patterns of biomarkers that differ significantly from those obtained from healthy subjects. The availability of fast, reproducible, and easy-to-apply analytical techniques that would allow identification of a large number of these analytes is thus highly desiderable since they may provide detailed information about the progression of a pathological process. From among the variety of methods so far applied for the determination of urinary metabolites, capillary electrophoresis, both in the capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes, represents a robust and reliable analytical tool widely used in this area. The aim of the present article is to focus the interest of the reader on recent applications of MEKC and CZE in the field of urinary biomarkers and to discuss advantages and/or limitations of each mode.

A micellar electrokinetic chromatographic method that allows simultaneous determination of both nucleotidase and transferase activities of cytosolic 5'-nucleotidase III is presented. This electrophoretic approach was successfully applied to human erythrocyte lysates to monitor the enzyme activities indicated above, using either physiological substrate or the nucleoside analogue 3'-azido-3'-deoxythymidine as the phosphate acceptor.

Tobacco smoke is involved in the pathogenesis of cardiovascular and respiratory diseases and also has a local toxic effect in the oral cavity. Low-aliphatic aldehydes, such as formaldehyde, acetaldehyde and acrolein, are among the main components of mainstream cigarette smoke and their local noxious and carcinogenic effects in the oral cavity and upper gastrointestinal tract are well-known. Although various studies have been performed so far to determine their content in cigarette smoke, none has included the direct measurement of these compounds in the saliva of smoking and nonsmoking subjects. Thus, in an attempt to verify whether typical chromatographic (high-performance liquid chromatography, HPLC) and/or electrophoretic (capillary electrophoresis, CE) techniques could be reliable methods for determining the levels of these analytes in human saliva, we submitted specimens obtained from a selected population of heavy, moderate, and nonsmoking subjects to HPLC and CE analyses. Both methods showed good reproducibility in terms of migration times and peak height and/or areas and had comparable linearity. Quantitative analyses performed on the specimens investigated evidenced a 3.5-fold increase of low-aliphatic aldehydes in saliva of nonsmoking subjects after they have smoked a single cigarette and a further 2-fold increase of these compounds in saliva of smokers with a daily consumption of 10 or more cigarettes.

The nasal lavage fluids (NLFs) from four subjects with acute sinusitis were analyzed to investigate the amount of proteins expressed in this pathology at the beginning of the event (day 1) and after 6 days of treatment with antibiotics and a nasal steroid spray. The protein identification was performed with capillary liquid chromatography-electrospray-quadrupole time of flight-(LC-ESI-Q-TOF)-mass spectrometry. The samples collected on the first day contained high-abundant plasma proteins, such as albumin and immunoglobulins, glandular serous cell proteins (lysozyme, lactoferrin, and polymeric immunoglobulin receptor), epithelial keratins, and inflammatory cell proteins (myeloperoxidase, IL-16, and IL-17E). After six days of therapy, the complexity of the proteome was reduced to plasma proteins and lysozyme with no inflammatory markers. The presence of hemoglobin, however, suggested that significant squamous metaplasia with breaches in the epithelial barrier, or nasal steroid-related bleeding, had occurred. The proteomic approach presented here allowed us to identify, in the high complexity of acute sinusitis nasal secretions, the proteins that respond to a pharmacological treatment and that could be suitable as markers of this pathology.

Among various biomarkers believed to behave as descriptors of the disease process in chronic obstructive pulmonary disease (COPD), urinary desmosines are commonly used for monitoring elastin degradation. Given the low concentrations of urinary desmosines, their quantitative determination in this biological matrix often requires preconcentration steps. To minimize both solute losses and effects of sample matrix, and to decrease data variability related to the above-mentioned manipulation processes, we have developed a capillary electrophoresis approach combined with laser-induced fluorescence (CE-LIF) detection system using urine samples not sub' mitted to any pretreatment procedure other than filtering the sample. Urines were hydrolyzed, derivatized with fluoresceine isothiocyanate (FITC) and endogenous desmosines were identified by addition of standard analytes and submitting to mass spectrometry (MS) analysis the material collected from micropreparative runs. The assay showed good linearity, reproducibility and precision, allowing to detect amounts of desmosines as low as 10 - 8 M (equivalent to 0.1 fmol on column). We conclude that CE-LIF technique is a highly sensitive method for detecting urinary desmosines.

Summary BackgroundProlidase deficiency is a rare genetic disorder for which a cure has not yet been found. Objectives To assess the effectiveness of apheresis exchange as a new therapeutic approach. Methods Apheresis exchanges were repeated monthly for four consecutive months, in parallel, on two patients, replacing prolidase-deficient red blood cells with normal filtered cells. Prolidase activity and urinary dipeptides were determined at regular intervals. Results The constant presence of active prolidase inside cells allowed a continuous, although partial, degradation of imidodipeptides, with a concomitant improvement of skin ulceration. Conclusions Apheresis exchange could be a reasonable way of obtaining a clinical improvement in these patients.

Owing to a series of interesting advantages, capillary electrophoresis (CE) is being widely used as an analytical tool for detecting enzymatic activities. On account of their resolving power, high speed, and small amount of sample required, several assays based on CE techniques have supported and/or replaced, during the past decade, traditional spectrophotometric or HPLC methods. In particular, a series of assays of practical utility have been designed for monitoring “in vitro” and “ex vivo”, the hydrolytic activity of proteinases of different origin involved in the development of human diseases. The aim of the present article is to focus the interest of the reader on some recent applications of different CE modes (capillary zone electrophoresis—CZE; micellar electrokinetic chromatography—MEKC and capillary isoelectric focusing—cIEF) in the field of proteolytic enzymes to underline the wide applicability of this methodology. The article presents examples of particular interest for which the advan...

This study looks at the impact of inflammation during the rehabilitation stage of strokes and its effect on neuro-functional recovery.This study investigated 94 patients suffering from strokes and admitted to rehabilitation. Anthropometric characteristics, serum proteins and inflammatory markers, plasma amino acids and neurofunction were all assessed.55.3% patients had an inflammatory status (Interleukin-6 = 19.24 ± 23.01 pg ml⁻¹ vs. 4.1 ± 1.6 pg ml⁻¹ for non-inflamed subjects (p 0.001). Inflammation was positively linked to positive proteins (alpha-1 globulin, p 0.02) and negatively linked to negative proteins (albumin, p 0.02; prealbumin, p 0.01; transferrin, p 0.05) of the acute-phase response. Inflammation was associated with low plasma concentrations of total amino acids. For the multiple logistic regression analysis, albumin (p 0.001) and body weight maintenance (p 0.001) were independent predictors of patient functional independence. Inflammation in dysphagic stroke (31.9%) patients was associated with more accentuated disability compared to non-inflamed dysphagics. The serum positive reactant alpha 1 globulin was the most powerful predictor of dysphagia severity (p 0.001). At discharge, dysphagia improvement was associated with improved acute-phase negative proteins.An inflammatory status may persist for most patients with strokes during the rehabiliation stage of the disease, its prevalence being higher in dysphagic compared to non-dysphagic subjects. The improvement in circulating albumin and body weight maintenance are predictors of neuro-function, even in dysphagic subjects.

Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (k(obs)) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (k(a)). A proflavine displacement assay was used to determine the deacylation rate constant (k(d)). Both Er and FE are good HNE inhibitors, showing a high k(a) and a low k(d). Because the number of turnovers per inactivation of Er was congruent with 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a k(d) only, whereas for FE, in addition to the k(d), an alkylation constant (k(2)) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.

Batten disease, or human late-infantile neuronal ceroid lipofuscinosis (LINCL) is a familiar progressive degenerative disease affecting children, caused by a deficiency of a lysosomal proteinase (tripeptidyl peptidase I, TPP-I) and characterized by the accumulation of autofluorescent storage bodies in the brain and other tissues of the body. Current methodology used to diagnose this disease needs to be improved in order to have less invasive techniques with higher resolution and shorter assay time. In this report, we discuss the potential merits of micellar electrokinetic chromatography as an excellent tool that requires minute samples but offers high resolution and a short running time for monitoring TPP-I activity in human and animal specimens.

A capillary isoelectric focusing (CIEF) method using bare fused-silica capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions in the pH 3.5-5.0 range has been developed for the identification of alpha1-antitrypsin (alpha1AT) phenotypes in human serum. This novel procedure was routinely applied to the study of serum samples of five controls whose alpha1AT phenotype was previously identified and of twelve subjects whose alpha1AT phenotype was unknown. The results obtained allowed us to confirm or identify the alpha1AT phenotype in all sera tested. This procedure seems particularly suitable for identification of alpha1AT variants associated with diseases of clinical relevance.

In order to use micellar electrokinetic chromatography to determine the proteolytic activity of different proteinases simultaneously present in physiological fluids, the technique must be able to separate mixtures of substrates with closely related structures. In an attempt to determine the best electrophoretic conditions for resolving six p-nitroanilide peptides used as synthetic substrates of the elastolytic enzymes (human neutrophil elastase, cathepsin G, Pseudomonas aeruginosa elastase) most commonly involved in pulmonary diseases, we investigated the efficiency of ionic and nonionic surfactants in achieving the separation of this complex mixture. The results presented here show that, of all the electrophoretic systems tested, 30 mM sodium tetraborate, pH 9.3, containing 25 mM Brij 35 as micellar agent offered the best performance; the separation efficiency of peptides is greater than that obtained with other reagents and all peaks are baseline resolved and unambiguously identifiable. Analysis of the micelle-solute interaction with the surfactants investigated allowed better definition of the mechanism involved in the distribution of these peptides to the micelles and identification of some structural features that determined the magnitude of the micelle peptide complex formation.