Plasma Proteomic Signatures in Early Chronic Obstructive Pulmonary Disease.

PURPOSE: Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and abnormal inflammatory response of the lungs to inhaled noxious particles or gases. We used a proteomic approach with 2-DE followed by MALDI TOF-MS analyses in order to identify potential biomarkers in the early stages of the disease: global initiative for chronic obstructive pulmonary disease (GOLD) stage mild and moderate. EXPERIMENTAL DESIGN: Blood plasma was collected from 43 patients with mild and moderate COPD as well as from 43 age- and sex-matched control subjects. Proteome analysis was based on 2D-Page followed by MALDI-TOF MS identifications. Validation was made on two significant proteins by western blotting. RESULTS: The analyses revealed 29 between-group differences in expressed spots, belonging to 20 unique proteins. These proteins are involved in inflammation (haptoglobin, Ig alpha-1 chain C), blood coagulation and complement pathways (prothrombin, complement 4-B, ApoH), oxidative stress (ceruloplasmin, vitamin D binding protein, and serotransferrin), and lipoprotein/lipid metabolism (apolipoprotein A-I, and apolipoprotein E). CONCLUSION AND CLINICAL RELEVANCE: These results indicate that specific proteomic signatures can be detected and useful in terms of treatment selection and in early COPD patient monitoring.

Proteomic analysis of human plasma and peripheral blood mononuclear cells in Systemic Lupus Erythematosus patients.

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a broad spectrum of clinical presentations and incompletely understood pathogenesis. This autoimmune disease is characterized by alterations in both the innate and adaptive immune system that lead to the loss of immunologic tolerance. In autoimmune diseases particularly in SLE, early diagnosis, flare or remission phases can be difficult to identify. Proteomics can help to find new therapeutic targets and it also could help to better understand the cellular mechanisms. The aim of this study was to observe the variations in plasma and Peripheral Blood Mononuclear Cells (PBMCs) proteome in order to increase our knowledge about pathogenesis and to find possible diagnostic markers and/or therapeutic targets for improving diagnosis and treatment. The comparative proteomic analyses showed that several proteins were differentially expressed in the PBMCs from SLE patients. Among these, PRDX2 may be used as candidate biomarker or target protein for further investigations. In plasma, we showed that plasma clusterin levels increased in SLE patients compared to healthy controls, but this increase is not statistically significant. These proteomic results provide suggestions for understanding the molecular mechanisms of SLE, as well as the physiological changes correlated with SLE disease.

Effects of Ramipril and Telmisartan on Plasma Concentrations of Low Molecular Weight and Protein Thiols and Carotid Intima Media Thickness in Patients with Chronic Kidney Disease.

Hypertension, a common feature in chronic kidney disease (CKD), is an independent risk factor for CKD progression and cardiovascular disease. Although inhibitors of the renin-angiotensin system (RAS) exert salutary effects on blood pressure control and proteinuria in CKD patients, their activity towards traditional and novel oxidative markers is largely unknown. We studied the effects of 6-month treatment with telmisartan versus a combination of telmisartan and ramipril on plasma concentrations of low molecular mass (LMW, including homocysteine and cysteine) and protein thiols (PSH) plasma concentration and their relationships with carotid intima media thickness (IMT), in 24 hypertensive CKD patients (age 60 ± 12 years, 8 females and 16 males). Pretreatment PSH concentrations were independently associated with IMT (r = -0.42, p = 0.039). Neither treatment affected plasma LMW thiols, in both reduced and total form. By contrast, both treatments increased PSH plasma concentrations and reduced IMT, although significant differences were only observed in the combined treatment group. Our results suggest that the beneficial effects of combined RAS inhibitor treatment on IMT in hypertensive CKD patients may be mediated by a reduction of oxidative stress markers, particularly PSH.

Plasma concentrations of transthyretin in older Sardinians including centenarians.

BACKGROUND AND AIMS: Plasma concentrations of transthyretin (TTR), a negative acute-phase protein, can be influenced by many factors including aging. Under physiological circumstances, TTR concentrations are very low in the fetus, increase slowly after birth up to the fifth decade and, then, decrease slowly. Some studies have shown sex-related differences up to about 70 years, when the differences disappear. The aim of this study was to evaluate the change in TTR concentrations in healthy males and females aged more than sixty, including numerous centenarians living in Sardinia, a large Italian island located in the Mediterranean Sea. METHODS: The study sample consisted of 211 healthy subjects grouped by age and sex (male/female ratio: 1:1). Plasma TTR was assessed using a non-competitive enzyme immunoassay (ELISA Assaypro LLC, prealbumin AssayMAX Human ELISA Kit). RESULTS: In subjects aged between 60 and 99 years, plasma TTR concentrations were higher compared to the reference ranges reported by CRM 470. Moreover, unlike other studies, sex-related differences in TTR concentrations were only observed in nonagenarians and centenarians. CONCLUSIONS: We hypothesize that there are TTR-related genetic differences between the Sardinian population and other Caucasian ethnic groups. Further studies and a larger sample are needed to confirm our hypothesis.

miRNA Stability in Frozen Plasma Samples.

MicroRNAs (miRNAs) represent a family of small non-coding ribonucleic acids that post-transcriptionally inhibits the expression of their target messenger RNAs (mRNAs), thereby acting as general gene repressors. In this study we examined the relative quantity and stability of miRNA subjected to a long period of freezing; we compared the stability of eight miRNAs in the plasma of five human healthy controls before freezing and after six and 12 months of storage at -80 °C. In addition, we examined the plasma frozen for 14 years and the amount of miRNA still available. Using a Life Technologies protocol to amplify and quantify plasma miRNAs from EDTA (Ethylene Diamine Tetraacetic Acid)-treated blood, we analyzed the stability of eight miRNAs, (miR-125b-5p, miR-425-5p, miR-200b-5p, miR-200c-3p, miR-579-3p, miR-212-3p, miR-126-3p, and miR-21-5p). The miRNAs analyzed showed a high stability and long frozen half-life.

Plasma Clusterin and Lipid Profile: A Link with Aging and Cardiovascular Diseases in a Population with a Consistent Number of Centenarians.

The role of Clusterin in attenuation of inflammation and reverse cholesterol transfer makes this molecule a potential candidate as a marker for cancer, cardiovascular disease, diabetes mellitus, and metabolic syndrome. In elderly subjects cardiovascular diseases represent the primary cause of death and different clinical studies have shown a positive correlation of these diseases with changes in the lipid pattern. This work aimed at evaluating the relationship between circulating clusterin and the biochemical parameters that characterize the lipid profile of a Sardinian population divided into five age groups including centenarians; the high frequency in Sardinia of these long-lived individuals gave us the opportunity to extend the range of the age groups to be analyzed to older ages and to better evaluate the changes in the lipid balance during ageing and its relationship with clusterin concentration in plasma. Our results showed that Clusterin concentration values of the youngest group were more similar with the centenarian's group compared to the other age groups, and a positive correlation arises with LDL. Furthermore given the high prevalence of cardiovascular diseases in the population examined and the association of Clusterin with these pathologies we evaluated Clusterin concentration variation in two groups with or without cardiovascular diseases. In presence of cardiovascular disease, Clusterin is significantly related to the most atherogenic components of lipid profile (total cholesterol and LDL), especially in women, suggesting its potential role in modulating cardiovascular metabolic risk factors.

Pre-analytical stability of the plasma proteomes based on the storage temperature.

BACKGROUND: This study examined the effect of storage temperature on the protein profile of human plasma. Plasma samples were stored for 13 days at -80°C, -20°C, +4°C and room temperature (20-25°C) prior to proteomic analysis. The proteomic comparisons were based on the differences of mean intensity values of protein spots between fresh plasma samples (named "time zero") and plasma samples stored at different temperatures. To better understand the thermally induced biochemical changes that may affect plasma proteins during storage we identified proteins with different expressions with respect to the time zero sample. RESULTS: Using two-dimensional electrophoresis followed by MALDI-TOF MS and /or LC-MS/MS 20 protein spots representing 10 proteins were identified with significant differences in abundance when stored at different temperatures. Our results, in agreement with various authors, indicate that during storage for a short period (13 days) at four different temperatures plasma proteins were more affected by degradation processes at +4°C compared to the other temperatures analysed. However, we founded that numerous protein spots (vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein A-I, apolipoprotein E, haptoglobin and complement factor B) decrease in abundance with increasing temperature up to 4°C, but at room temperature their intensity mean values are similar to those of time zero and -80°C. We hypothesize that these proteins are labile at 4°C, but at the same time they are stable at room temperature (20-25°C). Furthermore we have grouped the proteins based on their different sensitivity to the storage temperature. Spots of serum albumin, fibrinogen gamma chain and haptoglobin are more resistant to the higher temperatures tested, as they have undergone changes in abundance only at room temperature; conversely, other spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as they have undergone changes in abundance at all temperatures except at -80°C. CONCLUSIONS: Although there are many studies concerning protein stability of clinical samples during storage these findings may help to provide a better understanding of the changes of proteins induced by storage temperature.

Inferring gene networks: dream or nightmare?

Inferring gene networks is a daunting task. We here describe several algorithms we used in the Dialogue for Reverse Engineering Assessments and Methods (DREAM2) Reverse Engineering Competition 2007: an algorithm based on first-order partial correlation for discovering BCL6 targets in Challenge 1 and an algorithm using nonlinear optimization with winning performance in Challenge 3. After the gold standards for the challenges were released, the performance of alternative variants of the algorithms could be evaluated. The DREAM competition taught us some strong lessons. Amazingly, simpler methods performed in general better than more advanced, theoretically motivated approaches. Also, the challenges strongly showed that inferring gene networks requires controlled experimentation using a well-defined experimental design. Analyzing data obtained through merging many unrelated datasets indeed resulted in weak performances of all algorithms, while algorithms that explicitly took the experimental design into account performed best.