Elena Donadio

Serum levels of beta-trace protein and glomerular filtration rate--preliminary results.

The aim of this study was to evaluate the relationship between serum levels of beta-trace protein (BTP), a low molecular weight (MW) protein, and glomerular filtration rate (GFR). GFR and serum levels of BTP, and for comparison creatinine (Creat), cystatin C (Cys) and beta 2-microglobulin (beta 2M), were measured in 60 patients, with renal function ranging from normality to advanced renal failure. Serum levels of BTP progressively increased with the reduction of GFR. A good correlation was found between GFR and serum levels of BTP (r=0.918), Creat (r=0.932), Cys (r=0.937), and beta 2M (r=0.924). Furthermore, no statistically significant difference was found between BTP and Creat, Cys, beta 2M, as indicators of a moderate GFR impairment. These preliminary data indicate that BTP might be suitable as an indicator of GFR.

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands.

BackgroundProteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.ResultsThe best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.ConclusionsIn spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.

A proteomic profile of washing fluid from the colorectal tract to search for potential biomarkers of colon cancer

Washing fluid (WF) from the colon rectal tract after surgical resection might represent a first step in obtaining a mixture of proteins derived from the secretion of tumoral epithelial cells potentially involved in the pathological progression of tissue. In this study, we performed a proteomic analysis of colorectal WF to search for potential biomarkers of colon cancer. The outcome of this approach might open the possibility of using WF to screen for the precancerous and early stages of colorectal cancer (CRC). Samples of WFs were obtained during surgery from 35 patients submitted to colon resection for suspicious adenocarcinoma or carcinoma, while the respective controls were obtained by washing the healthy sections. WFs were immediately centrifuged, concentrated and trichloroacetic acid (TCA) was added to obtain protein pellets. After two-dimensional gel electrophoresis (2DE), the protein patterns of malignant samples were compared with respective normal samples. Forty-one protein spots were found to be differentially expressed exhibiting ≥2 fold-change of mean value spot intensities. After mass spectrometry, these protein spots collapsed into 38 different proteins. Interestingly, 19 of the differentially expressed proteins identified in the study corresponded to those suggested as being potential biomarkers of CRC. In accordance with the literature, these proteins showed the same direction of change (up or down for all proteins). Our results suggest that WF has the potential of being a method for the exploration of clinical samples for biomarker and drug target discovery.

A multidisciplinary approach to study a couple of monozygotic twins discordant for the chronic fatigue syndrome: a focus on potential salivary biomarkers.

BackgroundChronic Fatigue Syndrome (CFS) is a severe, systemic illness characterized by persistent, debilitating and medically unexplained fatigue. The etiology and pathophysiology of CFS remains obscure, and diagnosis is formulated through the patient’s history and exclusion of other medical causes. Thereby, the availability of biomarkers for CFS could be useful for clinical research. In the present study, we used a proteomic approach to evaluate the global changes in the salivary profile in a couple of monozygotic twins who were discordant for CFS. The aim was to evaluate differences of salivary protein expression in the CFS patient in respect to his healthy twin.MethodsSaliva samples were submitted to two-dimensional electrophoresis (2DE). The gels were stained with Sypro, and a comparison between CFS subject and the healthy one was performed by the software Progenesis Same Spot including the Analysis of variance (ANOVA test). The proteins spot found with a ≥2-fold spot quantity change and p<0.05 were identified by Nano-liquid chromatography electrospray ionization tandem mass spectrometry. To validate the expression changes found with 2DE of 5 proteins (14-3-3 protein zeta/delta, cyclophilin A, Cystatin-C, Protein S100-A7, and zinc-alpha-2-glycoprotein), we used the western blot analysis. Moreover, proteins differentially expressed were functionally analyzed using the Ingenuity Pathways Analysis software with the aim to determine the predominant canonical pathways and the interaction network involved.ResultsThe analysis of the protein profiles allowed us to find 13 proteins with a different expression in CFS in respect to control. Nine spots were up-regulated in CFS and 4 down-regulated. These proteins belong to different functional classes, such as inflammatory response, immune system and metabolism. In particular, as shown by the pathway analysis, the network built with our proteins highlights the involvement of inflammatory response in CFS pathogenesis.ConclusionsThis study shows the presence of differentially expressed proteins in the saliva of the couple of monozygotic twins discordant for CFS, probably related to the disease. Consequently, we believe the proteomic approach could be useful both to define a panel of potential diagnostic biomarkers and to shed new light on the comprehension of the pathogenetic pathways of CFS.

Search for peripheral biomarkers in patients affected by acutely psychotic bipolar disorder: a proteomic approach

Data on neurobiological mechanisms underlying mood disorders are elusive; the aetiology of such states is multifactorial, including genetic predisposition and environmental factors. Diagnosis is currently being made only on an interview-based methodology. Biological markers, which could improve the current classification, and in perspective, stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. We describe here a comparative proteomic analysis of peripheral lymphocytes from patients affected by acute psychotic bipolar disorder (PBD) (n = 15), major depressive episode (MDE) with no personal or family history of psychosis (n = 11), and a group of demographically matched healthy controls (HC) (n = 15). All patients were evaluated by means of Structured Clinical Interview for DSM-IV-Patient version (SCID-I-P), Positive and Negative Symptoms Scale (PANSS), Young Mania Rating Scale (YMRS), Hamilton Anxiety Rating Scale (HAM-A) and Hamilton Depression Rating Scale (HAM-D-17) questionnaires. Blood lymphocytes were obtained by gradient separation, and 2-DE was carried out on protein extracts. Significant differences in protein patterns among the three groups were observed. Thirty-six protein spots were found to be differentially expressed in patients compared to controls, which collapsed into 25 different proteins after mass spectrometry identification. Twenty-one of these proteins failed to discriminate between PBD and MDE, suggesting common signatures for these disorders. Nevertheless, after the western blot validation only two of the remaining proteins, namely LIM and SH3 domain protein1, and short-chain specific acyl-CoA dehydrogenase mitochondrial protein, resulted in being significantly upregulated in PBD samples suggesting additional mechanisms that could be associated with the psychotic features of bipolar disorder.

Serum albumin fragmentation in end-stage renal disease patients - a pilot study

Abstract Background: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. Methods: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. Results: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated Mr of 12.5–29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. Conclusions: These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood. Clin Chem Lab Med 2009;47:1373–9.

New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthins tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthins tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthins tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.

Parathyroid hormone and large related C-terminal fragments increase at different rates with worsening of renal function in chronic kidney disease patients. A possible indicator of bone turnover status?

BACKGROUND The intact parathyroid hormone (PTH) serum value has been the non-invasive biomarker of choice for the early diagnosis of renal bone disease in the chronic kidney disease (CKD) patient population. It has now been known that the intact PTH assay value is the sum of 1-84 PTH (true hypercalcemic PTH) and large C-terminal PTH fragments, mainly 7-84 PTH, a fragment with hypocalcemic hormone actions. AIM The aim of this study was to investigate the differences among the different functional stages of CKD in the following PTH parameters: intact PTH, 1-84 PTH, 7-84 PTH, and the ratio 1-84 PTH/7-84 PTH. GFR (clearance of 99mTc-DTPA) was measured in 164 (85 males and 79 females) adult CKD patients with different degrees of renal function impairment (serum creatinine 0.50 12.1 mg/dl, mean 2.00). PATIENTS AND METHODS Plasma concentrations of calcium, phosphate, 1-84 PTH and intact PTH were also measured. The value of 7-84 PTH was calculated as the difference between intact PTH and 1-84 PTH. The reduction of, GFR was accompanied by an increase of intact PTH, with a prevalent increase of 7-84 PTH over 1-84 PTH, resulting in a decrease of the ratio 1-84 PTH/7-84 PTH. RESULTS The values of 7-84 PTH showed a discrimination between Stages 1 and 2 (GFR > 60 ml/min ) and Stage 3 (GFR 30 60 ml/ min) CKD patient populations. In fact, 7-84 PTH was already significantly increased in patients at CKD Stage 3. The analysis of individual patients indicated that a low value (< 1.4) of the ratio 1-84 PTH/7-84 PTH, suggestive for low bone turnover, was already found in more than 20% of CKD Stage 3 patients. CONCLUSION The results of the present study demonstrate that the reduction in GFR is accompanied by a higher increase in 7-84 PTH with respect to 1-84 PTH, which suggests the possibility that bone metabolism and calcemic status are already reduced in patients with moderate renal failure (CKD Stage 3).

Osteopontin expression in healing wounds of horses and in human keloids.

REASONS FOR PERFORMING STUDY Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. OBJECTIVES To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. STUDY DESIGN Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. METHODS Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. RESULTS Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. CONCLUSIONS Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man.

Comparative proteomic analysis of malignant pleural mesothelioma evidences an altered expression of nuclear lamin and filament‐related proteins

Malignant mesothelioma is a neoplastic disease linked to asbestos exposure whose diagnosis is limited, so detection methods for an early diagnosis and treatment result essential. Here, we compared proteomic profiles of malignant pleural mesothelioma (MPM) and benign biopsies to search potential biomarkers useful in differential diagnosis.