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Elisa BELLEI

Personale tecnico amministrativo
sede ex-Scienze Biomediche Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

2023 - Research of Prostate Cancer Urinary Diagnostic Biomarkers by Proteomics: The Noteworthy Influence of Inflammation [Articolo su rivista]
Bellei, Elisa; Caramaschi, Stefania; Giannico, Giovanna A; Monari, Emanuela; Martorana, Eugenio; Reggiani Bonetti, Luca; Bergamini, Stefania
abstract

: Nowadays, in the case of suspected prostate cancer (PCa), tissue needle biopsy remains the benchmark for diagnosis despite its invasiveness and poor tolerability, as serum prostate-specific antigen (PSA) is limited by low specificity. The aim of this proteomic study was to identify new diagnostic biomarkers in urine, an easily and non-invasively available sample, able to selectively discriminate cancer from benign prostatic hyperplasia (BPH), evaluating whether the presence of inflammation may be a confounding parameter. The analysis was performed by two-dimensional gel electrophoresis (2-DE), mass spectrometry (LC-MS/MS) and Enzyme-Linked Immunosorbent Assay (ELISA) on urine samples from PCa and BPH patients, divided into subgroups based on the presence or absence of inflammation. Significant quantitative and qualitative differences were found in the urinary proteomic profile of PCa and BPH groups. Of the nine differentially expressed proteins, only five can properly be considered potential biomarkers of PCa able to discriminate the two diseases, as they were not affected by the inflammatory process. Therefore, the proteomic research of novel and reliable urinary biomarkers of PCa should be conducted considering the presence of inflammation as a realistic interfering element, as it could hinder the detection of important protein targets.

2023 - SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application [Articolo su rivista]
Isani, Gloria; Bellei, Elisa; Rudelli, Cecilia; Cabbri, Riccardo; Ferlizza, Enea; Andreani, Giulia
abstract

Abstract: In human and veterinary medicine, serum proteins are considered useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome which could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees, to suggest a panel of these proteins which could represent useful biomarkers for assessing the nutritional and health status of the colonies and finally to analyse them in different period of the year. Four apiaries were selected in the province of Bologna and bees analysed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph collected. The most represented bands obtained after 1D-SDS-PAGE were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of 12 proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. Two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis, and the second as a storage protein. Most of these proteins showed an increase from April to November mirroring the physiological changes of honeybees during the productive season. The current study suggested a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

2022 - Diagnostic proteomic markers to detect kidney diseases [Abstract in Rivista]
Ozben, T.; Bellei, E.; Monari, E.; Bergamini, S.; Ferrari, A.; Tomasi, A.
abstract

Objective Early detection of kidney disorders based on selective biomarkers could permit to diagnose patients at the initial stage of the disease, where the therapy is still possible to stop or prevent occurrence of advance disease. Urinary proteomics is primarily applied to the study of renal and urogenital tract disorders. Here are reported two distinct successful examples of this approach for the discovery of early urinary biomarkers of kidney-related dysfunctions: diabetic nephropathy (DN), a well-known complication of diabetes frequently leading to dialysis, and drug-induced nephrotoxicity, a possible condition caused by medication-overuse headache (MOH). Methods Urine samples were first concentrated and desalted. Subsequently, they were subjected to two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MS) for protein identification. Furthermore, some proteins were verified by Western Blot and ELISA test. Results In diabetes-related study, 11 differentially expressed proteins were detected (8 up-regulated and 3 down-regulated) in Type 2 Diabetic (T2D) and Type 2 Diabetic Nephropathy (T2DN) patients compared to the healthy control subjects. In MOH study, a total of 21 over-excreted proteins was revealed in urine of non-steroidal anti-inflammatory drugs (NSAIDs) and mixtures abusers versus controls. Particularly, 4 proteins were positively validated by immunoblotting and ELISA. Conclusion Urinary proteomics allows non-invasive assessment of renal diseases at an early stage by the identification of characteristic protein pattern.

2022 - Iron Speciation and Iron Binding Proteins in Arthrospira platensis Grown in Media Containing Different Iron Concentrations [Articolo su rivista]
Isani, Gloria; Niccolai, Alberto; Andreani, Giulia; Dalmonte, Thomas; Bellei, Elisa; Bertocchi, Martina; Tredici, Mario R.; Rodolfi, Liliana
abstract

Cyanobacteria are characterized by high iron content. This study investigated the effects of varying iron concentrations (1, 5, and 10 mg L−1) in the culture media on the biochemical composition and the iron bioaccumulation and speciation in Arthrospira platensis F&M‐C256. Iron content measured in biomasses varied from 0.35 to 2.34 mg g−1 dry weight depending on the iron concentration in the culture media. These biomasses can be considered of interest for the production of spirulina‐based supplements with low and high iron content. Iron speciation was studied using size exclusion chromatography followed by atomic absorption spectrometry and proteomic analysis. The role of C‐phycocyanin as an iron binding protein was also investigated. Overall, the present results provide a better understanding of iron metabolism in cyanobacteria and a foundation for further studies.

2022 - Looking for biomarkers of health status in the hemolymph of Apis mellifera [Poster]
Rudelli, Cecilia; Cabbri, Riccardo; Bellei, Elisa; Andreani, Giulia; Isani, Gloria
abstract

The demographic decline of honeybees (Apis mellifera) determines negative effects not only for agriculture and apiculture, but also for the ecosystems. For this reason, it is mandatory to understand the determinants causing the decline and to establish objective criteria to evaluate their health status. Proteins in the hemolymph may represent a source of valuables biomarkers to assess the health status of the hive; however, they are still poorly investigated. The aim of this study is to separate, to identify the most abundant proteins in the hemolymph, and to identify biomarkers of health status that could help beekeepers and veterinarians in the management and treatment of the hives. Four apiaries were chosen for this research, characterized by different management and location (A= hill, B and D= plain, C= apiary subjected to nomadism). In each apiary, three hives were selected and bees were collected in June, July, August, September, and October. Then 2 µL of hemolymph were withdrawn from each bee and the total proteins were determined by the Bradford method. The proteins of the hemolymph were separated using the SDS-PAGE electrophoresis and identified using mass spectrometry. Colony-level traits (areas covered with bees, brood, sealed brood and pollen) were also estimated. In the hemolymph of honeybees, 20 proteins were identified and among these, five proteins showed relevant variations. These are vitellogenin, apolipophorin, hexamerin, transferrin and prophenoloxidase. In particular, in October, the apiary C presented worst general conditions and a lower population associated with lower concentrations of total proteins, apolipoforin and vitellogenin. The apiary D, on the contrary, had the best trophic and health conditions, associated with higher concentrations of all the aforementioned five proteins. Thereby, it is possible to hypothesize that these proteins may be valuables biomarkers of health status, and it is worthy to continue their study.

2022 - Proteomics Disclose the Potential of Gingival Crevicular Fluid (GCF) as a Source of Biomarkers for Severe Periodontitis [Articolo su rivista]
Bellei, Elisa; Bertoldi, Carlo; Monari, Emanuela; Bergamini, Stefania
abstract

: Periodontal disease is a widespread disorder comprising gingivitis, a mild early gum inflammation, and periodontitis, a more severe multifactorial inflammatory disease that, if left untreated, can lead to the gradual destruction of the tooth-supporting apparatus. To date, effective etiopathogenetic models fully explaining the clinical features of periodontal disease are not available. Obviously, a better understanding of periodontal disease could facilitate its diagnosis and improve its treatment. The purpose of this study was to employ a proteomic approach to analyze the gingival crevicular fluid (GCF) of patients with severe periodontitis, in search of potential biomarkers. GCF samples, collected from both periodontally healthy sites (H-GCF) and the periodontal pocket (D-GCF), were subjected to a comparison analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A total of 26 significantly different proteins, 14 up-regulated and 12 down-regulated in D-GCF vs. H-GCF, were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The main expressed proteins were inflammatory molecules, immune responders, and host enzymes. Most of these proteins were functionally connected using the STRING analysis database. Once validated in a large scale-study, these proteins could represent a cluster of promising biomarkers capable of making a valuable contribution for a better assessment of periodontitis.

2022 - “Urinalysis, urinary proteome and metabolome of zoo-housed giraffes (Giraffa camelopardalis) through noninvasive sampling method”. [Relazione in Atti di Convegno]
Fasoli, Sabrina; Andreani, Giulia; Dondi, Francesco; Ferlizza, Enea; Bellei, Elisa; Sandri, Camillo; Laghi, Luca; Zhu, Chenglin; Isani, Gloria
abstract

The study of non-domestic animals withholds more difficulties compared to the domestic counterpart, thus using noninvasive techniques to collect biological samples might play an important role in assessing the health status of wild animals. 1 The present study established the reliability of urine sampling from the ground. A preliminary study was run with 10 urine samples of 10 cows (Bos taurus) housed in a dairy farming in Northern Italy. Urine samples, collected both in sterile cups and from the ground, were analyzed and compared. Results revealed no statistical differences in the variables investigated (p > 0.05, dipstick parameters and USG, protein quantification and UPC and protein electrophoresis), which proved the reliability of this noninvasive sampling method. This method was used for sampling 103 urine samples from 44 zoo-housed giraffes (Giraffa camelopardalis) of four Italian zoos. Urine samples were used to establish the urinalysis reference values in this species and to study the urinary proteome for the first time. The urinary reference values reported as median (lower and upper limit) were: urine specific gravity (USG), 1.030 (1006 - 1.049); urine total proteins (uTP), 17.58 (4.54 – 35.31) mg/dL; urine creatinine (uCr), 154.62 (39.59 – 357.95) mg/dL; urine protein: creatinine ratio (UPC), 0.11 (0.07 – 0.16). In giraffes, most urinary proteins had a low molecular mass (MM) and were present in low quantities. Proteomics disclosed fifteen different proteins, which were involved in the defense against microbes and in the ability of giraffes to concentrate urine. Albumin, lysozyme C, and ubiquitin were the most represented urinary proteins in giraffes. In addition, to define the urinary metabolome profile, 35 urine samples from 35 zoo-housed giraffes of five Italian zoos were used. Metabolomics allowed to identify and quantify 39 molecules and the most represented metabolites were hippurate, creatinine and phenylacetylglycine. This analysis provided information on physiological adaptations of giraffes. Besides, urinary metabolites were influenced by sex: urinary metabolome profile of female showed higher level of acetate, succinate, and lactate, conversely hippurate, phenylacetylglycine, and thymine were more concentrated in male urines. Similarly, the age affected the concentration of three urinary metabolites, namely formate, alanine, and valerate.

2021 - A Proteomic Analysis of Discolored Tooth Surfaces after the Use of 0.12% Chlorhexidine (CHX) Mouthwash and CHX Provided with an Anti-Discoloration System (ADS) [Articolo su rivista]
Bergamini, Stefania; Bellei, Elisa; Generali, Luigi; Tomasi, Aldo; Bertoldi, Carlo
abstract

Chlorhexidine (CHX) is considered the gold standard for the chemical control of bacterial plaque and is often used after surgical treatment. However, CHX employment over an extended time is responsible for side effects such as the appearance of pigmentations on the teeth and tongue; the discoloration effects are less pronounced when using a CHX-based mouthwash with added an anti-discoloration system (ADS). The aim of this study was to evaluate, using one- and two-dimensional gel electrophoresis combined with mass spectrometry, the possible proteomic changes induced by CHX and CHX+ADS in the supragingival dental sites susceptible to a discoloration effect. The tooth surface collected material (TSCM) was obtained by curettage after resective bone surgery from three groups of patients following a supportive therapy protocol in which a mechanical control was combined with placebo rinses or CHX or a CHX+ADS mouthwash. The proteomic analysis was performed before surgery (basal conditions) and four weeks after surgery when CHX was used (or not) as chemical plaque control. Changes in the TSCM proteome were only revealed following CHX treatment: glycolytic enzymes, molecular chaperones and elongation factors were identified as more expressed. These changes were not detected after CHX+ADS treatment. An ADS could directly limit TSCM forming and also the CHX antiseptic effect reduces its ability to alter bacterial cell permeability. However, Maillard's reaction produces high molecular weight molecules that change the surface properties and could facilitate bacterial adhesion.

2021 - A machine learning approach to study demographic alterations in honeybee colonies using SDS–PAGE fingerprinting [Articolo su rivista]
Cabbri, R.; Ferlizza, E.; Bellei, E.; Andreani, G.; Galuppi, R.; Isani, G.
abstract

Honeybees, as social insects, live in highly organised colonies where tasks reflect the age of individuals. As is widely known, in this context, emergent properties arise from interactions between them. The accelerated maturation of nurses into foragers, stimulated by many negative fac-tors, may disrupt this complex equilibrium. This complexity needs a paradigm shift: from the study of a single stressor to the study of the effects exerted by multiple stressors on colony homeostasis. The aim of this research is, therefore, to study colony population disturbances by discriminating overaged nurses from proper aged nurses and precocious foragers from proper aged foragers using SDS-PAGE patterns of haemolymph proteins and a machine-learning algorithm. The KNN (K Near-est Neighbours) model fitted on the forager dataset showed remarkably good performances (accu-racy 0.93, sensitivity 0.88, specificity 1.00) in discriminating precocious foragers from proper aged ones. The main strength of this innovative approach lies in the possibility of it being deployed as a preventive tool. Depopulation is an elusive syndrome in bee pathology and early detection with the method described could shed more light on the phenomenon. In addition, it enables countermeas-ures to revert this vicious circle.

2021 - Comparison of pregnenolone sulfate, pregnanolone and estradiol levels between patients with menstrually-related migraine and controls: an exploratory study [Articolo su rivista]
Rustichelli, Cecilia; Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Lo Castro, Flavia; Baraldi, Carlo; Tomasi, Aldo; Ferrari, Anna
abstract

Background Neurosteroids affect the balance between neuroexcitation and neuroinhibition but have been little studied in migraine. We compared the serum levels of pregnenolone sulfate, pregnanolone and estradiol in women with menstrually-related migraine and controls and analysed if a correlation existed between the levels of the three hormones and history of migraine and age. Methods Thirty women (mean age ± SD: 33.5 ± 7.1) with menstrually-related migraine (MM group) and 30 aged- matched controls (mean age ± SD: 30.9 ± 7.9) participated in the exploratory study. Pregnenolone sulfate and pregnanolone serum levels were analysed by liquid chromatography-tandem mass spectrometry, while estradiol levels by enzyme-linked immunosorbent assay. Results Serum levels of pregnenolone sulfate and pregnanolone were significantly lower in the MM group than in controls (pregnenolone sulfate: P = 0.0328; pregnanolone: P = 0.0271, Student’s t-test), while estradiol levels were similar. In MM group, pregnenolone sulfate serum levels were negatively correlated with history of migraine (R2 = 0.1369; P = 0.0482) and age (R2 = 0.2826, P = 0.0025) while pregnenolone sulfate levels were not age-related in the control group (R2 = 0.04436, P = 0.4337, linear regression analysis). Conclusion Low levels of both pregnanolone, a positive allosteric modulator of the GABAA receptor, and pregnenolone sulfate, a positive allosteric modulator of the NMDA receptor, involved in memory and learning, could contribute either to headache pain or the cognitive dysfunctions reported in migraine patients. Overall, our results agree with the hypothesis that migraine is a disorder associated with a loss of neurohormonal integrity, thus supporting the therapeutic potential of restoring low neurosteroid levels in migraine treatment.

2021 - Dehydroepiandrosterone Sulfate, Dehydroepiandrosterone, 5α-Dihydroprogesterone and Pregnenolone: Serum Analysis and Correlation Between Migraine and Non-headache Control Females [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Monari, Emanuela; Avallone, Rossella; Bellei, Elisa; Bergamini, Stefania; Tomasi, Aldo; Ferrari, Anna
abstract

Migraine is a very painful and disabling disorder of the nervous system (NS) affecting about 10% of the world's adult population, especially women and it is associated with a variety of comorbidities [1, 2]. Neuroactive steroids have pleiotropic actions on the NS. Alterations in their peripheral and central levels could be involved in the pathogenesis, still not fully understood, of migraine and its comorbidities [3]. The purpose of our exploratory study (approved by Modena Ethical Committee) was to determine serum levels of dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 5α-dihydroprogesterone (DHP) and pregnenolone (PREGNE) in women suffering from migraine without aura (n=30) and age-matched non-headache control females (n=30). The patients were enrolled at the Headache Centre of Modena; controls were patients’ contacts. Calibrators and serum samples were spiked with the ISs solution and treated to deplete proteins and phospholipids. The obtained extracts were evaporated to dryness, derivatized and analysed by RP-LC-ESI-MS/MS in MRM mode. Analyte’s levels were determined by interpolation on the regression curves, generated from the analyte quantifier ion peak area to the corresponding IS. Migraine women presented significantly lower levels of DHEAS, DHEA and DHP compared to controls (P<0.05) and the found concentrations negatively correlated with migraine history, and migraine days in the last three months (P< 0.05). These results parallel to our previous studies showing reduced serum levels of allopregnanolone and pregnenolone sulfate in migraine women [4,5]. The low serum levels found for both inhibitory and excitatory neurosteroids indicate that migraine women may suffer from inadequate neuroprotection, anti-inflammation activity and pain modulation. These deficits could represent the link between migraine and its various comorbidities. References [1] Headache classification Committee of the International Headache Society (IHS). Cephalalgia 2018, 38,1–211. [2] Yin JH., Lin YK., Yang CP., et al. Headache 2021. doi: 10.1111/head.14106. Online ahead of print. [3] Yilmaz, C.; Karali, K.; Fodelianaki, G.; et al. Front Neuroendocrinol 2019, 55, 100788. [4] Rustichelli, C.; Bellei, E.; Bergamini, S.; et al. Cephalalgia 2020, 40, 1355-1362. [5] Rustichelli, C.; Bellei, E.; Bergamini, S.; et al. J Head Pain 2021, 22, 13.

2021 - Dehydroepiandrosterone sulfate, dehydroepiandrosterone, 5α-dihydroprogesterone and pregnenolone in women with migraine: analysis of serum levels and correlation with age, migraine years and frequency [Articolo su rivista]
Rustichelli, Cecilia; Monari, Emanuela; Avallone, Rossella; Bellei, Elisa; Bergamini, Stefania; Tomasi, Aldo; Ferrari, Anna
abstract

Migraine is a very painful, disabling and extremely common disorder among the world's adult population, especially women, and it is associated with a variety of comorbidities. Neuroactive steroids exhibit pleiotropic actions on the nervous system. Alterations in their peripheral and central levels could be involved in the pathogenesis, still not fully understood, of migraine and its comorbidities. The purpose of our exploratory study was to determine and compare the serum levels of dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 5α-dihydroprogesterone (DHP) and pregnenolone (PREGNE) between women suffering from migraine without aura (MO group, n=30) and age-matched non-headache women as controls (C group, n=30). Correlations with age, migraine years and frequency were also analyzed. The patients were enrolled at a headache centre; controls were patients’ contacts. Calibrators and serum samples were spiked with the internal standards (ISs) solution and treated to deplete proteins and phospholipids. The obtained extracts were evaporated to dryness, derivatized and analysed by LC-MS/MS in multiple reaction monitoring mode. Analytes’ levels were determined by interpolation on the regression curves, generated from the analyte quantifier ion peak area to the corresponding IS. MO group presented significantly lower levels of DHEAS, DHEA and DHP compared to C group (P <0.05, Student’t test) and the neurosteroid levels negatively correlated with years of migraine and migraine days/3 months (P <0.05, linear regression analysis). These results parallel to previous studies showing reduced serum levels of allopregnanolone and pregnenolone sulfate in women with migraine. The low serum levels found for both excitatory and inhibitory neurosteroids suggested that women with migraine might suffer from inadequate neuroprotection, anti-inflammation activity and pain modulation. These deficits might underlie the migraine chronification process and represent the link between migraine and its various comorbidities.

2021 - Diagnostic proteomic biomarkers to detect kidney diseases [Abstract in Rivista]
Ozben, Tomris; Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; Pini, Luigi Alberto; Tomasi, Aldo
abstract

Urinary proteomics is primarily applied to the study of renal and urogenital tract disorders. Here are reported two distinct successful examples of this approach for the discovery of early urinary biomarkers of kidney­ related dysfunctions: diabetic nephropathy (DN), a well ­known complication of diabetes frequently leading to dialysis, and drug­induced nephrotoxicity, a possible condition caused by medication ­overuse headache (MOH). Early detection of kidney disorders based on selective biomarkers could permit to diagnose patients at the initial stage of the disease, where the therapy is still possible to stop or prevent occurrence of advance disease. Urine samples were first concentrated and desalted. Subsequently, they were subjected to two­-dimensional gel electrophoresis (2­DE) coupled to mass spectrometry (MS) for protein identification. Furthermore, some proteins were verified by Western blot and ELISA test. In diabetes-­related study, 11 differentially expressed proteins were detected (8 up­regulated and 3 down­regulated) in type 2 diabetic (T2D) and T2DN patients compared to the healthy control subjects. In MOH study, a total of 21 over­excreted proteins was revealed in urine of non­-steroidal anti­inflammatory drugs (NSAIDs) and mixtures abusers vs controls. Particularly, 4 proteins were positively validated by immunoblotting and ELISA. Urinary proteomics allows non­invasive assessment of renal diseases at an early stage by the identification of characteristic protein pattern.

2021 - Essential (Mg, Fe, Zn and Cu) and non-essential (Cd and Pb) elements in predatory insects (Vespa crabro and Vespa velutina): a molecular perspective [Articolo su rivista]
Andreani, Giulia; Ferlizza, Enea; Cabbri, Riccardo; Fabbri, Micaela; Bellei, Elisa; Isani, Gloria
abstract

The recent introduction of the Asian hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using gel filtration chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to a higher aerobic energy demand of the former species required to sustain foraging flight at higher speeds and to cover longer distances. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies.

2021 - Urinary proteomic profiles of prostate cancer with different risk of progression and correlation with histopathological features [Articolo su rivista]
Bergamini, S.; Caramaschi, S.; Monari, E.; Martorana, E.; Salviato, T.; Mangogna, A.; Balduit, A.; Tomasi, A.; Canu, P.; Bellei, E.
abstract

Prostate cancer (PCa) is the most common tumor in men with extremely variable outcome, varying from latent or indolent form to very aggressive behavior. High grade tumors, expansions exceeding the prostatic capsule into the surrounding soft tissues and spreading through lymph vascular channels, represent the most consistent unfavorable prognostic factors. However, accuracy in the prediction of the disease progression is sometimes difficult. Along with new molecular diagnostic techniques and more accurate histopathological approaches, proteomic studies challenge to identify potential biomarkers predictive of PCa progression. In our study we analyzed the urinary proteomes of 42 patients affected by PCa through two-dimensional electrophoresis associated with mass spectrometry. Proteomic profiles were correlated to histopathological features including pTNM stage and tumor differentiation in order to provide new promising markers able to define more accurately the PCa aggressiveness and driving new therapeutic approaches.

2021 - Urinary proteomics reveals promising biomarkers in menstrually related and post-menopause migraine [Articolo su rivista]
Bellei, Elisa; Bergamini, Stefania; Rustichelli, Cecilia; Monari, Emanuela; DAL PORTO, Michele; Fiorini, Alessandro; Tomasi, Aldo; Ferrari, Anna
abstract

Abstract: Migraine is an invalidating neuro-vascular disorder largely spread in the world population. Currently, its pathophysiology is not yet completely understood. The purpose of this study was to investigate the urinary proteome of women suffering from menstrually-related migraine (MM) and post-menopause migraine (PM) in comparison with non-headache women as controls, to search potential biomarkers of these migraine sub-types. Urine samples were analysed by mono-dimensional gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2DE) coupled to liquid chromatography-mass spectrometry (LC-MS/MS). Twenty-one urinary proteins were found significantly dysregulated in MM and PM (p<0.05). STRING Analysis database revealed interaction between 15 proteins, that resulted mainly involved in immune and inflammatory response. Seven of the most considerable proteins were further quantified by Western-blot: protein S100A8 (S10A8), up-regulated in MM, uromodulin (UROM), alpha-1-microglobulin (AMBP), gelsolin (GELS) and prostaglandin-H2 D-isomerase (PTGDS), over-expressed in PM, apolipoprotein A-I (APOA1) and transthyretin (TTHY), respectively down- and up-regulated in both migraineur groups vs controls. These candidate biomarkers might be involved in the neurophysiological network of MM and PM, thus helping to better understand the pathophysiology of these migraine forms. If validated in large-scale studies, this protein cluster could become a distinctive target for clinical applications in migraine diagnosis and treatment.

2020 - Heparin-induced lipoprotein precipitation apheresis in dyslipidemic patients: A multiparametric assessment [Articolo su rivista]
Merolle, L; Marraccini, C; Latorrata, A; Quartieri, E; Farioli, D; Scarano, L; Fasano, T; Bergamini, S; Bellei, E; Monari, E; Tomasi, A; Di Bartolomeo, E; Baricchi, R; Pertinhez, Ta
abstract

Low-density lipoprotein (LDL) apheresis (LA) selectively eliminates lipoproteins containing apolipoprotein B 100 (ApoB100) on patients affected by severe dyslipidemia. In addition to lowering lipids, LA is thought to exert pleiotropic effects altering a number of other compounds associated with atherosclerosis, such as pro- and anti-inflammatory cytokines or pro-thrombotic factors.

2020 - Identification of candidate proteomic markers in the serum of medication overuse headache patients: An exploratory study [Articolo su rivista]
Pellesi, Lanfranco; Guerzoni, Simona; Baraldi, Carlo; Cainazzo, Maria Michela; Pini, Luigi Alberto; Bellei, Elisa
abstract

The pathophysiological mechanism of medication overuse headache is uncertain; no distinctive markers have been described right now. The aim of this study was to conduct proteomic analyses on serum samples from patients with medication overuse headache and healthy individuals. Specifically, mono- (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to evaluate changes in serum proteins.

2020 - Proteomic serum profile in menstrual-related and post menopause migraine [Articolo su rivista]
Bellei, Elisa; Rustichelli, Cecilia; Bergamini, Stefania; Monari, Emanuela; Baraldi, Carlo; Lo Castro, Flavia; Tomasi, Aldo; Ferrari, Anna
abstract

The aim of this pilot study was to analyze the serum proteomic profile of women suffering from menstrual-related migraine (MM group, n = 15) and migraine in post-menopause (PM group, n = 15) in comparison with non-headache control females (C group, n = 15). Serum samples were subjected to two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) analysis for protein identification. Based on 2D-gel maps and PDQuest 2-D software, 13 differentially expressed spots, corresponding to 12 unique proteins identified by Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight/tandem mass spectrometry (LC-ESI-QToF-MS/MS), were detected in the MM and PM groups vs C group. Five inflammatory and regulatory of vascular integrity proteins (prothrombin, serum amyloid P-component, Ig kappa chain C region, apolipoprotein A-I, serum amyloid A-4 protein) were found deregulated in both MM and PM groups compared to C group; MM group showed the upregulation of other inflammatory protein fragments (inter-alpha-trypsin inhibitor heavy chain H4 and complement C4-A) compared to C group; PM group, in comparison with C group, displayed a noteworthy upregulation of transthyretin and other deregulated proteins (tetranectin, alpha-1-antitrypsin, haptoglobin, apolipoprotein A-IV) playing a role in anti-inflammatory and reparative processes. In conclusion, proteomic analysis was able to reveal differences in protein expression between migraine sufferers and non-headache women; as in other neurological diseases characterized by neuroinflammation, the serum proteome of migraine women presents an abundance of proteins indicative of cellular damage, oxidative stress and inflammation. This relevant inflammatory status, if confirmed in larger series, could represent a target for menstrual-related migraine treatment.

2020 - Serum levels of allopregnanolone, progesterone and testosterone in menstrually-related and postmenopausal migraine: A cross-sectional study [Articolo su rivista]
Rustichelli, Cecilia; Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Baraldi, Carlo; Castro, Flavia Lo; Tomasi, Aldo; Ferrari, Anna
abstract

Background: Reduced blood or cerebrospinal fluid levels of allopregnanolone are involved in menstrual cycle-linked CNS disorders, such as catamenial epilepsy. This condition, like menstrually-related migraine, is characterized by severe, treatment-resistant attacks. We explored whether there were differences in allopregnanolone, progesterone and testosterone serum levels between women with menstrually-related migraine (MM, n¼30) or postmenopausal migraine without aura who had suffered from menstrually-related migraine during their fertile age (PM, n¼30) and non-headache control women in fertile age (FAC, n¼30) or post-menopause (PC, n¼30). Methods: Participants were women with migraine afferent to a headache centre; controls were female patients’ acquaintances. Serum samples obtained were analyzed by HPLC-ESI-MS/MS. Results: In menstrually-related migraine and postmenopausal migraine groups, allopregnanolone levels were lower than in the respective control groups (fertile age and post-menopause) (p<0.001, one-way analysis of variance followed by Tukey-Kramer post-hoc comparison test) while progesterone and testosterone levels were similar. By grouping together patients with migraine, allopregnanolone levels were inversely correlated with the number of years and days of migraine/ 3 months (p 0.005, linear regression analysis). Conclusion: Decreased GABAergic inhibition, due to low allopregnanolone serum levels, could contribute to menstrually-related migraine and persistence of migraine after menopause. For the management of these disorders, a rise in the GABAergic transmission by increasing inhibitory neurosteroids might represent a novel strategy.

2020 - Urinary proteome and metabolome in dogs (Canis lupus familiaris): The effect of chronic kidney disease [Articolo su rivista]
Ferlizza, E.; Isani, G.; Dondi, F.; Andreani, G.; Vasylyeva, K.; Bellei, E.; Almeida, A. M.; Matzapetakis, M.
abstract

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands <65 kDa) proteinuria. Urine spectra acquired by NMR allowed the identification of 86 metabolites in healthy dogs, belonging to 49 different pathways mainly involved in amino acid metabolism, purine and aminoacyl-tRNA biosynthesis or tricarboxylic acid cycle. Seventeen metabolites showed significantly different concentrations when comparing healthy and CKD dogs. In particular, carnosine, trigonelline, and cis-aconitate, might be suggested as putative biomarkers of CKD in dogs. Significance: Urine is an ideal biological sample, however few proteomics and metabolomics studies investigated this fluid in dogs and in the context of CKD (chronic kidney disease). In this research, applying a multi-omics approach, new insights were gained regarding the molecular changes triggered by this disease in canine urinary proteome and metabolome. In particular, the involvement of the tubular component was highlighted, suggesting uromodulin, trigonelline and carnosine as possible biomarkers of CKD in dogs.

2020 - Urinary reference values and first insight into the urinary proteome of captive giraffes [Articolo su rivista]
Sabrina, Fasoli; Giulia, Andreani; Francesco, Dondi; Enea, Ferlizza; Bellei, Elisa; Gloria, Isani
abstract

Abstract. Urinalysis is widely recognized to be a useful tool in routine health investigation since it can diagnose numerous pathologies. Considering the paucity of knowledge concerning giraffes, urine from 44 giraffes (Giraffa camelopardalis) (18 males and 26 females, from 3 months of age to 21 years of age) underwent routine urinalysis, 1D-electrophoresis and protein identification using mass spectrometry, with the aim of identifying the urinary reference values and the urine proteome. Urine specific gravity (USG), urine total proteins (uTP), urine creatinine (uCr) and the urine protein:creatinine ratio (UPC) reference values, reported as median, and lower limit (LL) and upper limit (UL), were 1.030 (1006 - 1.049), 17.58 (4.54 – 35.31) mg/dL, 154.62 (39.59 – 357.95) mg/dL and 0.11 (0.07 – 0.16), respectively. Mass spectrometry together with electrophoresis revealed a pattern of common urinary proteins; albumin, lysozyme C and ubiquitin were the most represented proteins in the giraffe urine. It has been hypothesized that these proteins could act as a defense against microbes. Moreover, in giraffes, urinalysis could be a valid tool for gauging renal function and physiological status changes.

2019 - Allopregnanolone serum levels in female migraineurs [Abstract in Rivista]
Rustichelli, C.; Bellei, E.; Bergamini, S.; Monari, E.; Lo Castro, F.; Baraldi, C.; Cainazzo, M. M.; Tomasi, A.; Ferrari, A.
abstract

Background: Migraine and epilepsy are similar brain disorders in many aspects and for both, a neuronal hyperexcitability has been hypothesized. Cyclic changes in ovarian hormones are involved in exacerbating both migraine and epilepsy during perimenstrual period, leading to menstrually-related migraine and catamenial epilepsy, respectively. Ovarian hormones and derived neurosteroids can regulate important functions in neurons and glial cells in the brain; in particular, progesterone reduces seizure susceptibility partly through its conversion to allopregnanolone, a potent positive allosteric modulator of the GABA-A receptor [1]. In spite of their neuroprotective potential [2], the role of neurosteroids in migraine has not been thoroughly investigated. Therefore, we determined serum levels of testosterone, progesterone and allopregnanolone in three groups: women suffering from menstrually-related migraine (n=30), post-menopausal women suffering from migraine without aura (n=30) and non-headache control females (n=20). Methods: The enrolled migraineurs were patients afferent to the Headache Centre of Modena University Hospital; the control females were friends or relatives of the above patients. All women gave their written consent and the Ethical Committee of the Province of Modena approved the study. The fasting blood specimens were processed and then analyzed by HPLC-ESI-MS/MS. Results: Testosterone and progesterone levels were significantly higher in both non-headache control females and women suffering from menstrually-related migraine compared to post-menopausal women suffering from migraine without aura (P <0.005, t-test). Conversely, serum allopregnanolone levels were significantly lower in both women suffering from menstrually-related migraine (0.051 ng/mL; SD: 0.018) and post-menopausal women suffering from migraine without aura (0.025 ng/mL; SD: 0.013), compared to non-headache control females (0.078 ng/mL; SD 0.036, P <0.005, t-test). Conclusion: Women suffering from migraine presented low serum levels of allopregnanolone, a neurosteroid that modulates GABAergic inhibition. Consequently, the reduced GABAergic inhibition could inadequately protect women suffering from migraine against inflammatory and algogenic stimuli. In particular, it could contribute to the severity and poor response to treatments of migraine attacks. According to our preliminary results, a raise in the GABAergic transmission achieved by drugs increasing the biosynthetic pathway of inhibitory neurosteroids or the use of synthetic analogs could represent a possible novel therapeutic strategy for migraine management. [1] Meletti S., et al J. Neurochem. 2018; 147:275-284. [2] Reddy D.S., et al. Trends Pharmacol Sci. 2016;37:543-561.

2019 - Comparative proteomic analysis between the gingival crevicular fluid and the corresponding periodontal pocket: a preliminary study [Articolo su rivista]
Bertoldi, Carlo; Bergamini, Stefania; Ferrari, Monica; Lalla, Michele; Bellei, Elisa; Spinato, Sergio; Tomasi, Aldo; Monari, Emanuela
abstract

Introduction The aim of this study was to compare the proteomic profile of periodontal pocket tissues with that of corresponding gingival crevicular fluid (GCF), and to search for similarities in their proteomic profile. Material and Methods Four patients suffering from moderate or severe chronic periodontitis, needing surgical periodontal treatment, were selected. Immediately before the periodontal surgery, GCF samples were taken by means of filter paper strips positioned in the gingival sulcus correspondent to periodontal pockets. Then, periodontal pocket tissue, harvested during surgery, was adequately stored for proteomic analyses. Results Using an image analysis software for proteomic data, we found almost the same protein expression profile in GCF and pocket tissue from each patient. Accordingly, in our patients we found no statistically significant correlation between the quantitative proteomic profile of GCF and pocket tissue. Only one band (that of K immunoglobulin) resulted statistically different between GCF and pocket tissue proteome in all patients (p=0.008). Conclusions The protein network of the periodontal pocket does not influence significantly the GCF protein network. The periodontal pocket and the GCF are similar as far as the proteomic networks are concerned, but the GCF does not seem suitable to study on the pathogenesis of periodontal disease.

2019 - Evaluation of potential cardiovascular risk protein biomarkers in high severity restless legs syndrome [Articolo su rivista]
Bellei, E.; Bergamini, S.; Monari, E.; Tomasi, A.; Koseoglu, M.; Topaloglu Tuac, S.; Ozben, S.
abstract

Restless legs syndrome (RLS) is a common sensorimotor disorder that, in case of severe symptoms, can be very distressing and negatively interfere with quality of life. Moreover, increasing evidences associate RLS with higher risk of cerebrovascular and cardiovascular disease (CVD). The purpose of this study was to quantify two proteins, previously identified by proteomics and potentially linked with CVD risk, namely kininogen-1 (KNG1) and alpha-1-antitrypsin (A1AT), in primary RLS patients at high severity grade (HS-RLS) in comparison to healthy control subjects. Proteins were quantified through enzyme-linked immunosorbent assay (ELISA) in plasma samples from 14 HS-RLS patients and 15 control individuals. The two groups were closely matched for age and gender. The expression level of KNG1 resulted significantly higher (p < 0.001), while A1AT was significantly decreased (p < 0.05) in HS-RLS patients compared to controls, confirming the relationship between these proteins and the disease severity. Furthermore, in patients group the association between the protein concentrations and the following parameters was further evaluated: age, disease onset and diagnosis, scores obtained from the RLS rating scales (Epworth Sleepiness Scale, Pittsburgh Sleep Quality Index, Beck Depression Inventory) and smoking habit. All the considered variables resulted independent of protein levels, so the disease can be reasonably considered the main cause of protein changes. As emerged from the literature, high levels of KNG1 and low amounts of A1AT seem to be related with a highest probability to develop CVD. Consequently, these proteins may be reliable candidate biomarkers of CVD risk in patients with RLS at high severity grade.

2019 - Exploration of candidate serum biomarkers potentially related to the chronic pain condition in Medication-overuse headache [Articolo su rivista]
Pellesi, Lanfranco; Bellei, Elisa; Guerzoni, Simona; Michela Cainazzo, Maria; Baraldi, Carlo; Monari, Emanuela; Pini, Luigi Alberto
abstract

Background Medication Overuse Headache (MOH) is a prevalent and disabling disorder resulting from the overuse of analgesic drugs, triptans or other acute headache medications. In previous proteomic studies, several proteins have been found at high concentrations in the urine of MOH patients and in the serum of rats with neuropathic pain. The aim of this study was to compare the serum levels of lipocalin-type Prostaglandin D2 synthase (L-PGDS), Vitamin D-binding protein (VDBP), apolipoprotein E (APOE) and apolipoprotein A1 (APOA1) in MOH patients and healthy individuals, further exploring their relationship with cutaneous pain thresholds (CPTs) in the territories innervated by the trigeminal nerve. Methods 69 MOH patients and 42 age- and sex-matched healthy volunteers were enrolled in the study. Von Frey-like filaments were applied to the skin territories innervated by the trigeminal nerve, to determine the CPTs. L-PGDS, VDBP, APOE and APOA1 were quantified in the serum by Enzyme-linked Immunosorbent Assay (ELISA). Clinical and laboratory data were collected. Comparisons between MOH patients and healthy individuals were performed using independent t test or χ2 test. To correlate serum proteins with CPTs, Pearson correlation coefficient or Spearman's rank correlation coefficient were used. Results CPTs were lower among MOH patients. L-PGDS, VDBP and APOE had significantly different serum concentrations between groups (p < 0.01), but no correlation was found with CPTs. APOA1 serum concentrations did not differ between patients and healthy individuals. Conclusions L-PGDS, VDBP and APOE had abnormal serum levels in MOH patients, confirming their alteration in some conditions of chronic headache and neuropathic pain. The in-depth study of target proteins represents a promising approach for a better understanding of MOH, as well as the detection of candidate biomarkers for chronic headache or the risks associated with overuse medications.

2019 - Proteomic serum profile of female migraineurs [Abstract in Rivista]
Bellei, E; Bergamini, S; Monari, E; Rustichelli, C; Baraldi, C; Lo Castro, F; Tomasi, A; Ferrari, A
abstract

Background: Migraine is considered a complex disease, a variable disorder of nervous system function that has a genetic background, yet the final phenotypic outcome largely depends on the individual’s environment and lifestyle. In particular, there is a clear relationship between menstruation cycle and the onset of migraine. In fact, over 50% of migraine women suffer from perimenstrual attacks, that are more serious, lasting and resistant to the treatment than non-menstrual migraine attacks [1]. We hypothesized that serum proteome analysis could help to identify potential biomarkers of menstrually-related migraine (MM) and post-menopausal migraine (PMM). Methods: We analyzed and compared the serum proteomic profile of three groups: women suffering from MM (n=15), post-menopausal women suffering from migraine without aura (n=15) and non-headache control females (n=14). The enrolled migraineurs were patients afferent to the Headache Centre of Modena University Hospital; the control females were friends or relatives of the above patients. All women gave their written consent and the Ethical Committee of Modena approved the study. Serum samples obtained from each study participant were subjected to bi-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MS) analysis for protein identification. The 2D-gel maps were examined by the PDQuest software, to detect the differentially expressed protein spots between the different groups [2]. Results: A total of 13 significantly different protein spots were revealed in migraine women compared to controls. Of these proteins, most (n=10) resulted increased in migraineurs vs controls, while only 3 proteins were decreased. Specifically, the greater expression differences involved the up-regulation of transthyretin in PMM and the down-regulation of apolipoprotein A1 in MM. Other proteins, such as prothrombin, serum amyloid P-component and Ig-k-chain C region, were found significantly over-expressed in migraine sufferers in comparison to controls, while one spot, recognized as serum amyloid A-4 protein, resulted decreased. Conclusion: The serum proteome of migraine women showed proteins characteristic of cell damage, oxidative stress and lipoperoxidation, as well as acute phase proteins and inflammation markers. This pilot study demonstrates the ability of proteomics to reveal differences in protein expression between women suffering from MM and post-menopausal women suffering from migraine without aura against non-headache women. Further analysis will be carried out to expand and confirm these preliminary results. [1] Calhoun A.H. Headache 2018; 58:626-630. [2] Bellei E., et al. Amino Acids 2011; 40:145-156.

2018 - Discovery of restless legs syndrome plasmatic biomarkers by proteomic analysis [Articolo su rivista]
Bellei, Elisa; Monari, Emanuela; Ozben, Serkan; Koseoglu Bitnel, Mesrure; Topaloglu Tuac, Selma; Tomasi, Aldo; Bergamini, Stefania
abstract

Objectives: Restless legs syndrome (RLS) can lead to severe clinical consequences, thus negatively impacts on patients’ overall health and quality of life. Nevertheless, the pathophysiology of RLS is still unclear, resulting in underestimate, incorrect, or ignored diagnosis and in limited management and treatment. The aim of this study was to compare the plasma proteome of RLS patients and healthy controls, in the search of diagnostic biomarkers related to the disease severity. Materials and Methods: Two-dimensional gel electrophoresis coupled with liquid chromatography-mass spectrometry was employed to analyze plasma samples of 34 patients with primary RLS, divided into two subgroups according to the disease severity: MMS group (mild-moderate symptoms) and HS group (severe and very severe symptoms), and 17 age- and sex-matched control subjects. Sleep quality, daytime sleepiness, and the level of depression were also evaluated. Results: We identified eight upregulated spots, corresponding to five unique proteins, in both RLS group vs. controls (alpha-1B-glycoprotein, alpha-1-acid glycoprotein 1, haptoglobin, complement C4-A, and immunoglobulin kappa constant); five increased spots, consistent with three unique proteins, only in HS-RLS (kininogen-1, immunoglobulin heavy constant alpha 1, and immunoglobulin lambda constant 2); one downregulated spot in both patient's groups (complement C3) and another one only in HS-RLS (alpha-1-antitrypsin). Conclusions: The significantly different plasma proteins detected in RLS were mainly associated with inflammation, immune response, and cardiovascular disorders. Particularly, the gradual increasing in immunoglobulins could be indicative of the disease severity and evolution. Accordingly, these proteins may represent a valid set of useful biomarkers for RLS diagnosis, progression and treatment.

2018 - Serum changes of apolipoproteins in Medication Overuse Headache (MOH) [Abstract in Rivista]
Pellesi, Lanfranco; Bellei, Elisa; Baraldi, Carlo; Guerzoni, Simona; Monari, Emanuela; Pini, Luigi Alberto
abstract

Background Medication Overuse Headache (MOH) is a prevalent and disabling disorder resulting from the overuse of analgesic drugs, triptans or other acute headache medications. Previous proteomic studies have identified some altered proteins, including different forms of apolipoproteins, which are probably associated with the chronic painful symptom and its consequences. The aim of the study was to explore the relationship between cutaneous pain thresholds, Zung Self- Rating Depression Scale (ZUNG-D) scores, Leeds Dependence Questionnaire (LDQ) scores and serum levels of apolipoprotein A1 (APOA1) and apolipoprotein E (APOE) in patients with MOH. Methods 69 patients with MOH and 42 healthy volunteers as control group were enrolled in the study between September 2016 and January 2018. To investigate skin sensitivity, Von Frey-like filaments were applied sequentially to the skin territories innervated by the divisions of the trigeminal nerve, to determine cutaneous pain thresholds. APOA1 and APOE, previously identified as potential biomarkers candidates for the pathophysiology of chronic pain, were quantified in the serum by Enzyme-linked Immunosorbent Assay (ELISA). Results Cutaneous pain thresholds were lower among patients with MOH than healthy controls. Serum APOE was significantly lower in patients with MOH, compared to healthy volunteers (p < 0.01), but no differences were found concerning serum APOA1 (Fig. 1). In patients with MOH, serum APOE was positively related to Body Mass Index, albumin and uric acid, whereas serum APOA1 was positively related to creatinine. Serum APOA1 and APOE did not have any relationship with cutaneous pain thresholds, ZUNG-D scores, LDQ scores and other clinical or laboratory parameters. Conclusions Serum APOE is significantly altered in patients with MOH compared to controls, but it is apparently not correlable with any aspect of the disease. APOE may play a role in the pathophysiology of MOH and the consequences associated with medication overuse; further studies are needed to deepen this finding. Ethics approval This study was performed following the Helsinki Declaration principles and approved by the local Ethical Committee (prot. 2073).

2018 - Studio degli effetti della LDL aferesi in pazienti affetti da ipercolesterolemia familiare [Abstract in Rivista]
Merolle, L; Latorrata, A; Marraccini, C; Farioli, D; Scarano, L; Di Bartolomeo, E; Bergamini, S; Bellei, E; Monari, E; Fasano, T; Baricchi, R; Pertinhez, T. A.
abstract

La LDL-aferesi selettiva è un trattamento che riduce radicalmente l'LDL nei pazienti affetti da ipercolesterolemia familiare (FH) e al contempo va a modificare i livelli di altri composti associati al fenomeno dell'aterosclerosi.

2018 - Urinary proteomics in biomarker discovery of kidney-related disorders: Diabetic nephropathy and drug-induced nephrotoxicity in chronic headache [Articolo su rivista]
Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; Pini, Luigi Alberto; Tomasi, Aldo; Ozben, Tomris
abstract

Objective Urinary proteomics is primarily applied to the study of renal and urogenital tract disorders. Here are reported two distinct successful examples of this approach for the discovery of early urinary biomarkers of kidney-related dysfunctions: diabetic nephropathy (DN), a well-known complication of diabetes frequently leading to dialysis, and drug-induced nephrotoxicity, a possible condition caused by medication-overuse headache (MOH). Early detection of kidney disorders based on selective biomarkers could permit to diagnose patients at the initial stage of the disease, where the therapy may be suspended or prevent disease advancement. Methods Urine samples were first concentrated and desalted. Subsequently, they were subjected to two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MS) for protein identification. Furthermore, some proteins were verified by Western blot and ELISA test. Results In diabetes-related study, 11 differentially expressed proteins were detected (8 up-regulated and 3 down-regulated) in type 2 diabetic (T2D) and T2DN patients compared to the healthy control subjects. In the MOH study, a total of 21 over-excreted proteins were revealed in urine of non-steroidal anti-inflammatory drugs (NSAIDs) and mixtures abusers vs controls. Particularly, 4 proteins were positively validated by immunoblotting and ELISA. Conclusion Urinary proteomics allows non-invasive assessment of renal diseases at an early stage by the identification of characteristic protein pattern.

2018 - Validation of prostate cancer biomarkers and inflammation: a proteomic study [Relazione in Atti di Convegno]
Ozben, T; Bergamini, S; Bellei, E; Cuoghi, A; Bianchi, G; Tomasi, A
abstract

Analysis of serum proteome to investigate possible confounding paramenters in the discrimination between prostate cancer nad benign prostatic hyperplasia

2017 - Serum protein changes in a rat model of chronic pain show a correlation between animal and humans [Articolo su rivista]
Bellei, Elisa; Vilella, Antonietta; Monari, Emanuela; Bergamini, Stefania; Tomasi, Aldo; Cuoghi, Aurora; Guerzoni, Simona; Manca, Letizia; Zoli, Michele; Pini, Luigi Alberto
abstract

In previous works we showed the overexpression of some proteins in biological fluids from patients suffering chronic pain. In this proteomic study we analysed serum from a rat model of neuropathic pain obtained by the chronic constriction injury (CCI) of sciatic nerve, at two time intervals, 2 and 5 weeks after the insult, to find proteins involved in the expression or mediation of pain. Sham-operated and CCI rats were treated with saline or indomethacin. Two weeks after ligation, we identified three serum proteins overexpressed in CCI rats, two of which, alpha-1-macroglobulin and vitamin D-binding protein (VDBP), remained increased 5 weeks post-surgery; at this time interval, we found increased levels of further proteins, namely apolipoprotein A-I (APOA1), apolipoprotein E (APOE), prostaglandin-H2 D-isomerase (PTGDS) and transthyretin (TTR), that overlap the overexpressed proteins found in humans. Indomethacin treatment reversed the effects of ligation. The qPCR analysis showed that transcript levels of APOA1, APOE, PTGDS and VDBP were overexpressed in the lumbar spinal cord (origin of sciatic nerve), but not in the striatum (an unrelated brain region), of CCI rats treated with saline 5 weeks after surgery, demonstrating that the lumbar spinal cord is a possible source of these proteins.

2016 - Characterization of equine amniotic fluid by a proteomic approach [Abstract in Atti di Convegno]
Isani, G; Ferlizza, E; Cuoghi, A; Bellei, E; Monari, E; Bianchin Butina, B; Castagnetti, C
abstract

This study succesfully applied SDS-PAGE coupled to MS for the identification of the most abundant proteins in equine AF.

2016 - Identification of the most abundant proteins in equine amniotic fluid by a proteomic approach [Articolo su rivista]
Isani, Gloria; Ferlizza, Enea; Cuoghi, Aurora; Bellei, Elisa; Monari, Emanuela; Bianchin Butina, Barbara; Castagnetti, Carolina
abstract

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein identification. Mean AF protein concentration was 1.96 ± 1.12 g/L. Thirty-four proteins were successfully identified by MS and subsequently categorised according to Gene Ontology (GO). Twelve proteins (e.g. fibronectin, lumican, thrombospondin and fibulin) belonged to or interacted with the extracellular matrix (ECM) playing an important role in the development of foetal tissues. Most of the remaining proteins were classified as transport (e.g. albumin, major allergen Equ c1 and alpha-fetoprotein) delivering nutrients, ions and lipids essential for foetal growth and development. Among these proteins, major allergen Equ c1 is widely studied in human medicine because it induces Ig-E mediated type I allergic reaction. The absence of immunoglobulins in equine AF was also confirmed.

2016 - Influence of two different Varroa treatments on the most abundant proteins of honey bee hemolymph [Poster]
Ferlizza, E; Cabbri, R; Andreani, G; Bellei, E; Cuoghi, A; Monari, E; Isani, G
abstract

Evaluation of the effect of different techniques on vitellogenin and other abundant hemolymph proteins to obtain successful wintering of the colonies

2016 - Proteomic validation of biomarkers for discrimination of benign and malign prostatic hyperplasia [Abstract in Rivista]
Ozben, T; Bergamini, S; Bellei, E; Cuoghi, A; Bianchi, G; Tomasi, A
abstract

Use of proteomic techniques for the identification and validation of discriminating protein biomarkers in prostate cancer and BPH.

2015 - A proteomics study in cats affected by chronic kidney disease: identification of putative biomarkers [Abstract in Rivista]
Ferlizza, E; Campos, A; Cuoghi, A; Bellei, E; Monari, E; Dondi, F; Almeida, Am; Isani, G
abstract

Application of proteomic techniques for the identification of putative biomarkers of nephropathy in cats affected by chronic kidney disease.

2015 - Analysis of protein expression in periodontal pocket tissue: a preliminary study [Articolo su rivista]
Monari, Emanuela; Cuoghi, Aurora; Bellei, Elisa; Bergamini, Stefania; Lucchi, Andrea; Tomasi, Aldo; Cortellini, Pierpaolo; Zaffe, Davide; Bertoldi, Carlo
abstract

The periodontal disease is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. The proteomic profile and related molecular conditions of pocket tissue in periodontally-affected patients are not reported in literature. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. Proteins were separated using two-dimensional gel electrophoresis and identified by liquid chromatography/mass spectrometry. After identification, four proteins were selected for subsequent Western Blot quantitation both in pathological and healty tissues.

2015 - Erratum to: Proteomic analysis of protein extraction during hemofiltration with on-line endogenous reinfusion (HFR) using different polysulphone membranes [JMater Sci:MaterMed, 25, (2014) 2691-2698, DOI 10.1007/s10856-014-5290-5] [Articolo su rivista]
Monari, E.; Cuoghi, A.; Bellei, E.; Bergamini, S.; Caiazzo, M.; Aucella, F.; Loschiavo, C.; Corazza, L.; Palladino, G.; Sereni, L.; Atti, M.; Tomasi, A.
abstract

2015 - New horizon in dialysis depuration: Characterization of a polysulfone membrane able to break the 'albumin wall' [Articolo su rivista]
Cuoghi, Aurora; Caiazzo, Marialuisa; Monari, Emanuela; Bellei, Elisa; Bergamini, Stefania; Sereni, L; Aucella, F; Loschiavo, C; Atti, M; Tomasi, Aldo
abstract

The uremic syndrome is attributed to the progressive retention of a large number of toxins, which under normal conditions are excreted by the healthy kidneys. Standard dialytic membranes do not purify middle-high molecular weight toxins. Haemodiafiltration with endogenous reinfusion coupled with a highly permeable membrane could break the limit of the 'albumin wall' improving the dialytic depuration without loss of important nutrients. The aim of this study was to evaluate the performance of a new polysulfone membrane, Synclear 0.2, to remove uremic molecules. Surface Enhanced Laser Desorption Ionization-Time of Flight was employed to evaluate the proteomic profile of ultrafiltrate and Electrospray Ionization-Quadruple-ToF coupled with on-chip elution was used for proteins identification. A high and specific permeability for middle-high molecular weight molecules was revealed by mass spectrometry for the investigated membrane. The identified proteins are mostly uremic toxins: their relative abundance, estimated in the ultrafiltrate by exponentially modified protein abundance index, showed a high purification efficiency of the new membrane when compared with conventional ones. In conclusion, Synclear 0.2, used as convective membrane in hemodiafiltration with endogenous reinfusion treatment, permits to break the 'albumin wall', clearing middle-high molecular weight uremic toxins, improving the dialytic treatment purification efficiency.

2015 - Proteomic analysis of proteins adsorbed by resin cartridge filter during hemodiafitration with online endogenous reinfusion. [Abstract in Rivista]
Monari, Emanuela; Bergamini, Stefania; Cuoghi, Aurora; Bellei, Elisa; Solano, Francesco; Bruni, Francesco; Ozben, Tomris; Tomasi, Aldo
abstract

BACKGROUND-AIM Background: Lupus nephritis (LN) is kidney inflammation caused by systemic lupus erythematous (SLE), that lead to end stage renal disease and consequently to dialytic therapy. Inflammation mediators over-expression play a key role in disease initiation and progression. Immuno-complexes and/or autoantibodies deposition in the kidney induce cytokines production in renal resident cells, which may further amplify inflammatory processes. Hemodiafiltration with Endogenous Reinfusion (HFR) dialysis treatment with super high flux membrane Synclear 02 (SUPRA) is a dialytic method, which combines the diffusion and convection processes with adsorption by a resin cartridge filter. Proteomic approach was applied for protein separation and identification in order to evaluate the quality of proteins retained by resin bed during dialytic treatment. METHODS Methods: Plasma and ultrafiltrate (UF) samples of three patients with LN, treated with SUPRA HFR (Bellco, Italy), were collected at 15 and 235 min of two different dialytic sessions. The utilized cartridges, containing styrenic resin, were opened and the proteins kept by the resin were eluted. Gel electrophoresis was used to separate protein content before protein identification by ESI-QTOF-MS (Electrospray Ionization-Quadrupole Time-of-Flight-Mass Spectrometry) analysis RESULTS Results: The comparison of proteomic profiles of plasma, UF and eluted samples demonstrate the removal of several protein species by the resin bed. ESI-QTOF analysis allowed to identify several biomarker of kidney injury, such as: Retinol binding protein 4, Neutrophil gelatinase-associated lipocalin (NGAL), Prostaglandin-H2 D-isomerase, Cystatin- C, Serotransferrin, Alpha-1-acid glycoprotein (A1AG1), Transthyretin and several fragments of Immunoglobulins. Moreover, Beta-2-glycoprotein 1 (APO-H), involved in antiphospholipid syndrome, a disorder that manifests clinically as recurrent venous or arterial thrombosis, was identified. CONCLUSION Conclusions: The proteomic approach was used in this study to evaluate the performance of styrenic resin to retain proteins implicated in the LN pathogenesis and pathophisiology. The treatment with SUPRA-HFR demonstrate to be suitable to reduce inflammatory status, uremic toxin level and antiphospholipid syndrome in LN patients.

2015 - Proteomic research of proteins involved in pain expression in an animal model of chronic pain [Articolo su rivista]
Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Cuoghi, Aurora; Zoli, Michele; Tomasi, Aldo; Cainazzo, Maria Michela; Guerzoni, Simona; Pini, Luigi Alberto
abstract

nd

2015 - Proteomics in the clinic: the search for biomarkers [Abstract in Atti di Convegno]
Tomasi, A; Bellei, E; Bergamini, S; Cuoghi, A; Monari, E; Ozben, T
abstract

Biomarkers are molecules that exist naturally in the body; they can help to predict or reflect the presence of a disease, the relapse risk of the disease, and/or response to treatment.

2015 - Radical improvement of signs and symptoms in systemic lupus erythematosus when treated with hemodiafiltration with endogenous reinfusion dialysis [Articolo su rivista]
Solano, Francesco Giuseppe; Bellei, Elisa; Cuoghi, Aurora; Caiazzo, Marialuisa; Bruni, Francesco
abstract

Lupus nephritis is one of the most serious complications of systemic lupus erythematosus (SLE). In the kidney immune complexes (ICs) and autoantibodies activate mesangial cells that secrete the cytokines that further amplify inflammatory processes. We present a case of a 42-year-old woman presented Lupus Nephritis, with periods of exacerbation of SLE, with necrotic-like skin lesions, psoriatic arthritis without skin psoriasis, purpura to lower limb, petechial rash, joint pain, fever, eyelid edema with bilateral conjunctival hyperemia and itching When she was subjected to Hemodiafiltration with Endogenous Reinfusion (HFR) dialysis treatment with super high flux membrane Synclear 02 (SUPRA treatment), fever and joint pain was reduced immediately, subsequently all of her skin damages are reduced and she gradually decreased quantity of prednisone and immunosuppressor per die until completely suspend. As well known that SUPRA treatment remove cytokine from blood ; moreover was used the High-performance liquid chromatography coupled with Quadrupole Time-of-flight Mass Spectrometer (HPLC-QTOF-MS) for identification of proteins captured by resin bed during a dialysis session of the patient. With this technique was identified several biomarker of kidney injuries, uremic toxins, fragments of Immunoglobulins, antigen involved in anti-phospholipid syndrome and a new marker (α-defensin) that correlate significantly with disease activity. The removal of these different proteins can explain the improvement in the patient’s symptoms and the normalization of her LES, confirming that SUPRA are a suitable technique for LN treatment.

2015 - The effect of chronic kidney disease on the urine proteome in the domestic cat (Felis catus) [Articolo su rivista]
Ferlizza, E.; Campos, A.; Neagu, A.; Cuoghi, Aurora; Bellei, Elisa; Monari, Emanuela; Dondi, F.; Almeida, A. M.; Isani, G.
abstract

Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was con- ducted on 4–12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P &lt; 0.01). 2-DE was es- sential in fractionation of the complex urine proteome, producing a reference map that included 20 proteins. Cauxin was the most abundant protein in urine of healthy cats. Several protease inhibitors and trans- port proteins that derive from plasma were also identified, including alpha-2-macroglobulin, albumin, transferrin, haemopexin and haptoglobin. There was differential expression of 27 spots between healthy and CKD samples (P &lt; 0.05) and 13 proteins were unambiguously identified. In particular, increased ex- pression of retinol-binding protein, cystatin M and apolipoprotein-H associated with decreased expression of uromodulin and cauxin confirmed tubular damage in CKD cats suggesting that these proteins are can- didate biomarkers.

2015 - The importance of inflammation in the search of prostate cancer biomarkers [Poster]
Bergamini, S; Bellei, E; Monari, E; Cuoghi, A; Reggiani Bonetti, L; Borelli, F; Sighinolfi, C; Bianchi, G; Ozben, T; Tomasi, A
abstract

The importance of inflammation in the search of prostate cancer biomarkers by proteomics.

2015 - Validation of potential candidate biomarkers of drug-induced nephrotoxicity and allodynia in medication-overuse headache [Articolo su rivista]
Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; Cuoghi, Aurora; Tomasi, Aldo; Guerzoni, Simona; Ciccarese, Michela; Pini, Luigi Alberto
abstract

Medication-overuse headache (MOH) is a chronic disorder that results from the overuse of analgesics drugs, triptans or other acute headache compounds. Although the exact mechanisms underlying MOH remain still unknown, several studies suggest that it may be associated with development of "central sensitization", which may cause cutaneous allodynia (CA). Furthermore, the epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity. The aim of this work was to confirm and validate the results obtained from previous proteomics studies, in which we analyzed the urinary proteome of MOH patients in comparison with healthy non-abusers individuals.

2014 - A 2DE map of the urine proteome in the cat: effect of Chronic Kidney Disease [Relazione in Atti di Convegno]
Ferlizza, E; Campos, A; Cuoghi, A; Bellei, E; Monari, E; Dondi, F; Almeida, Am; Isani, G
abstract

The aim of this study was to produce a preliminary map of the urine proteome of healthy cats (Felis catus) and to compare it with the proteome of cats affected by chronic kidney disease.

2014 - Analisi tissutale proteomica della tasca parodontale. Uno studio pilota Periodontal pocket tissue analysis using proteome. A pilot study [Articolo su rivista]
Chiara, Pellacani; Monari, Emanuela; Zaffe, Davide; Cuoghi, Aurora; Bellei, Elisa; Andrea, Lucchi; Bergamini, Stefania; Tomasi, Aldo; Bertoldi, Carlo
abstract

Obbiettivo: Scopo dello studio è analizzare nello stesso soggetto, in siti in cui non erano rilevabili batteri parodontopatogeni, il tessuto inteprossimale, sia associato alla tasca parodontale sia sano, al fine di determinare un quadro proteico associabile al danno parodontale. Materiali & Metodi: Nello studio sono stati inclusi quindici soggetti sistemicamente sani, affetti da moderata-avanzata parodontite cronica, che presentavano almeno un difetto intraosseo prossimo ad un analogo sito senza danno parodontale clinicamente evidente. I pazienti sono stati trattati mediante terapia resettiva. Durante la fase chirurgica i tessuti associati alla lesione parodontale e quelli clinicamente sani sono stati prelevati per l’analisi proteomica. Risultati: Confrontando i profili proteici relativi al danno parodontale con quelli clinicamente sani, sono state identificate 19 proteine differentemente espresse. In particolare, in tutti i pazienti 8 proteine sono risultate sovra-espresse nel tessuto patologico (Anexina A2- ANX A2; Actina citoplasmatica 1 (spot 14 e 15)- ACTB; Anidrasi carbonica 1 - CAH1; Anidrasi carbonica 2- CAH2; Ig catena Kappa regione C (spot 17 e 18)- IGKC e flavina reduttasi- BLVRB) mentre 11 proteine sono risultate sotto-espresse (Tropomiosina catena -4- TPM3; proteina  14-3-3 - 1433S; proteina / 14-3-3 - 1433Z; -enolasi - ENOA; Heat shock proteina -1 (spot 5 e spot 6) - HSPB1; Triosofosfatoisomerasi - TPIS; Perossiredoxina-1 - PRDX1; Proteina epidermica legante acidi grassi - FABP5; Proteina S100-A9 - S10A9 e Galectina -7 - LEG7). Conclusioni: Dai dati preliminari ottenuti risulta evidenziata l’espressione differenziale, tra tessuto clinicamente sano e relativo al danno parodontale, di proteine che possono giocare un ruolo importante nella prevenzione del danno cellulare da stress, nella mediazione delle risposte immunitarie, nonché nei meccanismi di rigenerazione tissutale. Lo studio del profilo profilo proteomico del tessuto della tasca parodontale potrebbe essere cruciale sia per la conoscenza della patogenesi che per la terapia della malattia parodontale. Objective: To analyze in the same subject, in sites where no periodontopathogenic bacteria were detectable, pocket-associated and neighboring healthy interproximal tissues to qualify proteins associated with the periodontal damage. Matherials & Methods: Fifteen healthy patients, affected by moderate to advanced chronic periodontitis and presenting at least one intrabony defect and a neighboring not-damaged interproximal site were enrolled. Patients underwent osseous resective surgery. During surgery pocket-associated and clinically healthy tissues were harvested for proteomic analyses. Results: In both pocket-associated and clinically healthy tissues, nineteen differently expressed proteins were successfully identified. In particular, 8 proteins (Annexin A2; Actin cytoplasmic 1 (2 spots); Carbonic anhydrase 1; Carbonic anhydrase 2; Ig kappa chain C region (2 spots) and Flavinreductase) were over-expressed, while 11 proteins (Tropomyosin alpha-4 chain; 14-3-3 protein sigma; 14-3-3 protein zeta/delta; Alpha-enolase; Heat shock protein ß-1 (2 spots); Triosophosphateisomerase; Peroxiredoxin-1; Fatty acid-binding protein-epidermal; Protein S100-A9 and Galectin-7) were under-expressed in the pathological tissue of all patients. Conclusions: The preliminary data indicate differentially expression of proteins that may play important roles in the prevention of cellular damage by stress, in mediating the immune response as well as in tissue regeneration. The proteomic profile study of pocket tissue would be crucial both to appreciate the pathogenesis and the therapy of periodontitis.

2014 - Evaluation of proteomic profile in menstrual-related migraine [Abstract in Rivista]
Cuoghi, Aurora; Monari, Emanuela; Bellei, Elisa; Bergamini, Stefania; Tomasi, Aldo; Ferrari, Anna
abstract

nd

2014 - Evalutation of proteomic profile in menstrually-related migraine [Abstract in Rivista]
Cuoghi, A; Monari, E; Bellei, E; Bergamini, S; Tomasi, A; Ferrari, A.
abstract

Valutazione del profilo proteomico (sierico e urinario) nelle donne con emicrania correlata alle mestruazioni

2014 - Inflammation: an important parameter in the search of prostate cancer biomarkers [Articolo su rivista]
Bergamini, Stefania; Bellei, Elisa; REGGIANI BONETTI, Luca; Monari, Emanuela; Cuoghi, Aurora; Francesco, Borelli; Sighinolfi, Maria Chiara; Bianchi, Giampaolo; T., Ozben; Tomasi, Aldo
abstract

Background A more specific and early diagnostics for prostate cancer (PCa) is highly desirable. In this study, being inflammation the focus of our effort, serum protein profiles were analyzed in order to investigate if this parameter could interfere with the search of discriminating proteins between PCa and benign prostatic hyperplasia (BPH). Methods Patients with clinical suspect of PCa and candidates for trans-rectal ultrasound guided prostate biopsy (TRUS) were enrolled. Histological specimens were examined in order to grade and classify the tumor, identify BPH and detect inflammation. Surface Enhanced Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (SELDI-ToF-MS) and two-dimensional gel electrophoresis (2-DE) coupled with Liquid Chromatography-MS/MS (LC-MS/MS) were used to analyze immuno-depleted serum samples from patients with PCa and BPH. Results The comparison between PCa (with and without inflammation) and BPH (with and without inflammation) serum samples by SELDI-ToF-MS analysis did not show differences in protein expression, while changes were only observed when the concomitant presence of inflammation was taken into consideration. In fact, when samples with histological sign of inflammation were excluded, 20 significantly different protein peaks were detected. Subsequent comparisons (PCa with inflammation vs PCa without inflammation, and BPH with inflammation vs BPH without inflammation) showed that 16 proteins appeared to be modified in the presence of inflammation, while 4 protein peaks were not modified. With 2-DE analysis, comparing PCa without inflammation vs PCa with inflammation, and BPH without inflammation vs the same condition in the presence of inflammation, were identified 29 and 25 differentially expressed protein spots, respectively. Excluding samples with inflammation the comparison between PCa vs BPH showed 9 unique PCa proteins, 4 of which overlapped with those previously identified in the presence of inflammation, while other 2 were new proteins, not identified in our previous comparisons. Conclusions The present study indicates that inflammation might be a confounding parameter during the proteomic research of candidate biomarkers of PCa. These results indicate that some possible biomarker-candidate proteins are strongly influenced by the presence of inflammation, hence only a well-selected protein pattern should be considered for potential marker of PCa.

2014 - P-cresolo solfato e rischio cardiovascolare nel paziente trapiantato [Abstract in Atti di Convegno]
Ligabue, G; Granito, M; Tomasi, A; Cuoghi, A; Bellei, E; Cappelli, G
abstract

Il p-Cresol-Solfato è una tossina uremica che, provenendo esclusivamente dall'apporto dietetico, incrementa i livelli ematici in rapporto al grado di insufficienza renale e correla con l'aumento delle complicanze cardiovascolari

2014 - Proteomic analisys of protein extraction during hemofiltration with on-line endogenous reinfusion (HFR) using different polysulphone membranes [Articolo su rivista]
Monari, Emanuela; Cuoghi, Aurora; Bellei, Elisa; Bergamini, Stefania; Marialuisa, Caiazzo; Filippo, Aucella; Carmelo, Loschiavo; Luca, Corazza; Giuseppe, Palladino; Luisa, Sereni; Mauro, Atti; Tomasi, Aldo
abstract

In end-stage renal disease patients, extracor- poreal dialytic therapy is not able to prevent the accumu- lation of toxins related to the uremic syndrome, a severe complication that increases morbidity and mortality rate. In this paper, hemoFiltration with on-line Reinfusion (HFR) architecture is used to evaluate the effect of a more per- meable membrane on the extraction of medium–high molecular weight molecules. The aim of this study was to compare two polysulphone membranes for convective chamber: polyphenylene High Flux (pHF) and polyphen- ylene Super High-Flux (pSHF). Fourteen patients were subjected to HFR with pHF and pSHF membranes and ultra filtrate (UF) samples were collected to evaluate molecular weight cut-off (MWCO) and to identify extracted proteins. Furthermore, image analysis software was used in order to evaluate change in protein extraction during the dialysis. The quantification of four proteins by immunoassay dem- onstrates a higher permeability of pSHF membrane. Two- dimensional electrophoresis (2-DE) gels showed, for both Electronic supplementary material The online version of this article (doi:10.1007/s10856-014-5290-5) contains supplementary material, which is available to authorized users. M. Emanuela (&amp;) ? C. Aurora ? B. Elisa ? B. Stefania ? T. Aldo Department of Diagnostic, Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Via del Pozzo, 71-41124 Modena, Italy e-mail: emanuela.monari@unimore.it C. Marialuisa ? C. Luca ? P. Giuseppe ? S. Luisa ? A. Mauro Scientific Affairs, Bellco s.r.l, Mirandola, Modena, Italy A. Filippo IRCCS Hospital CSS, San Giovanni Rotondo, Foggia, Italy L. Carmelo Division of Nephrology and Dialysis, Legnago, Verona, Italy membranes, the greater number of protein spots at 235 min. Some of the identified proteins, involved in nephropathic disease complications, were compared to assess differences in extraction during dialytic treatment by PDQuest analysis. UF proteomic analysis demonstrated a different behavior for the two membranes; pHF membrane was more permeable at the beginning of HFR treatment (15 min), while pSHF membrane at the end of treatment (235 min). Proteomic analysis is a suitable approach to investigate the behavior of different membranes during dialysis. Results indicated that pSHF membrane offers the higher permeability, and showed higher efficiency in removal of middle molecules related to uremic syndrome.

2014 - Proteomic profile of retained proteins from hemodiafiltration with on-line endogenous reinfusion (SUPRA) cartridge. [Abstract in Rivista]
Cuoghi, Aurora; Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; Tomasi, Aldo; Atti, Mauro; Caiazzo, Marialuisa; Palladino, Giuseppe; Bruni, Francesco
abstract

nd

2014 - Validation of prostate cancer biomarkers and inflammation: a proteomic study [Abstract in Atti di Convegno]
Bergamini, S; Bellei, E; Monari, E; Cuoghi, A; Reggiani Bonetti, L; Borelli, F; Sighinolfi M., C; Bianchi, G; Ozben, T; Tomasi, A
abstract

Validation of prostate cancer biomarkers and inflammation by a proteomic approach

2013 - Detection of predictive urinary biomarkers of nephropathy in type 1 and type 2 diabetes by proteomic analysis [Abstract in Rivista]
Bellei, Elisa; Cuoghi, Aurora; Monari, Emanuela; Bergamini, Stefania; Ligabue, Giulia; Cappelli, Gianni; Ozben, Tomis
abstract

Background: Nephropathy associated with diabetes is a severe complication that cause slow kidneys deterioration, leading to end-stage renal disease. Renal involvement during diabetes mellitus may affect all the structural components of the kidneys, causing functional and organic alterations frequently associated with inflammatory processes, that give rise to multiple clinical manifestations. Currently, despite rapid research progress, predictors able to assess prospectively and with high precision the risk to develop diabetic nephropathy (DN) are still lacking. Methods: The aim of this project was to identify differences in urinary protein excretion, both in type 1 (T1D) and type 2 diabetic (T2D) patients, in comparison with healthy control subjects. Ninety diabetic patients were recruited and divided in 3 groups (for each diabetes type), according to the level of albuminuria: normoalbuminuric, with microalbuminuria (MA) and with overt proteinuria. Second void morning urine samples were collected and centrifuged to remove cell debris and contaminations. Urinary proteins were separated by twodimensional electrophoresis (2-DE) and identified by mass spectrometry analysis (MS). Results: Comparing the patients proteomic profiles with those of normal subjects, firstly we noted a significant increase of alpha-1-antitrypsin and albumin, also in the form of numerous fragments, in urine of diabetic subjects. Particularly, statistical analysis and spot quantification by PDQuest image software revealed several proteins differentially expressed in diabetes condition. Some proteins resulted increased in urine of both T1D and T2D patients with MA, such as transthyretin, apolipoprotein-A1 and transferrin, while the majority of the overexcreted proteins were found in T2D patients with proteinuria, e.g. vitamin-D-binding protein, protein AMBP, zinc-alpha-2- glycoprotein, fetuin-A and ganglioside GM2 activator. Conclusions: This protein pattern might represent a potential tool for a better understanding of DN and could help to identify patients at increased risk of renal disease progression. Therefore, in diagnostic field, 2-DE and MS proteomic analysis could be a suitable approach to discover early and predictive biomarkers of DN in urine of diabetic patients.

2013 - Discovery by a proteomic approach of possible early biomarkers of drug-induced nephrotoxicity in medication-overuse headache [Articolo su rivista]
Bellei, Elisa; Monari, Emanuela; Cuoghi, Aurora; Bergamini, Stefania; Guerzoni, Simona; Ciccarese, Michela; Ozben, T.; Tomasi, Aldo; Pini, Luigi Alberto
abstract

BACKGROUND: Medication-overuse headache (MOH) is a chronic headache condition that results from the overuse of analgesics drugs, triptans, or other antimigraine compounds. The epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity, particularly in chronic patients. The aim of this work was to confirm and extend the results obtained from a previous study, in which we analyzed the urinary proteome of 3 MOH patients groups: non-steroidal anti-inflammatory drugs (NSAIDs), triptans and mixtures abusers, in comparison with non-abusers individuals (controls). METHODS: In the present work we employed specialized proteomic techniques, namely two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS), and the innovative Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS), to discover characteristic proteomic profiles associated with MOH condition. RESULTS: By 2-DE and MS analysis we identified 21 over-excreted proteins in MOH patients, particularly in NSAIDs abusers, and the majority of these proteins were involved in a variety of renal impairments, as resulted from a literature search. Urine protein profiles generated by SELDI-TOF-MS analysis showed different spectra among groups. Moreover, significantly higher number of total protein spots and protein peaks were detected in NSAIDs and mixtures abusers. CONCLUSIONS: These findings confirm the presence of alterations in proteins excretion in MOH patients. Analysis of urinary proteins by powerful proteomic technologies could lead to the discovery of early candidate biomarkers, that might allow to identify MOH patients prone to develop potential drug overuse-induced nephrotoxicity.

2013 - Non-bacterial protein expression in periodontal pockets by proteome analysis [Articolo su rivista]
Bertoldi, Carlo; Bellei, Elisa; Chiara, Pellacani; Davide, Ferrari; Andrea, Lucchi; Aurora, Cuoghi; Bergamini, Stefania; Pierpaolo, Cortellini; Tomasi, Aldo; Zaffe, Davide; Monari, Emanuela
abstract

Objectives: To compare the proteomic profile of inter-proximal pocket tissues with inter-proximal healthy tissues in the same subject to reveal proteins associated with periodontal disease in sites where periodontopathogenic bacteria were not detectable. Methods: Twenty-five healthy patients, with moderate-to-advanced chronic periodontitis and presenting with at least one intra-bony defect next to a healthy inter-proximal site were enrolled. The periodontal defects were treated with osseous resective surgery, and the flap design included both the periodontal pockets and the neighbouring inter-proximal healthy sites. Pocket-associated and healthy tissues were harvested for proteomic analyses. Results: Fifteen proteins were differently expressed between pathological and healthy tissues. In particular, annexin A2, actin cytoplasmic 1, carbonic anhydrase 1 & 2; Ig kappa chain C region (two spots) and flavinreductase were overexpressed, whereas 14-3-3 protein sigma and zeta/delta, heat-shock protein beta -1 (two spots), triosephosphateisomerase, peroxiredoxin-1, fatty acid-binding protein-epidermal, and galectin-7 were underexpressed in pathological tissue. Conclusions: The unbalanced functional network of proteins involved could hinder adequate tissue response to pathogenic noxa. The study of periodontal pocket tissue proteomic profile would be crucial to better understand the pathogenesis of and the therapeutic strategies for periodontitis.

2013 - Proteomic Analysis of PTCH1+/- Fibroblast Lysate and Conditioned Culture Media Isolated from the Skin of Healthy Subjects and Nevoid Basal Cell Carcinoma Syndrome Patients. [Articolo su rivista]
Ponti, Giovanni; Bertazzoni, G; Pastorino, L; Monari, Emanuela; Cuoghi, Aurora; Bergamini, Stefania; Bellei, Elisa; Benassi, Luisa; Azzoni, Paola; Petrachi, Tiziana; Magnoni, Cristina; Pellacani, Giovanni; Loschi, P; Pollio, A; Witkowski, Am; Tomasi, Aldo
abstract

Background. The pathogenesis underlying the increased predisposition to the development of basal cell carcinomas (BCCs) in the context of Gorlin-Goltz syndrome is linked to molecular mechanisms that differ from sporadic BCCs. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age and present with tumors of non-sun-exposed skin. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and nonmutated fibroblasts. Results. Proteomic analysis was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. 12 protein cluster peaks, >5 kDa, had significant differences in their peak intensities between PTCH1+ and PTCH1- subject groups. We detected a strongly MMP1 overexpression in PTCH1+ fibroblasts obtained from NBCCS patients with respect to healthy donors. Conclusion. Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense versus nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies and to develop novel experimental molecular tailored therapies based on these druggable targets.

2013 - Proteomic analysis of PTCH1+/- fibroblast lysate and conditioned culture media isolated from the skin of healthy subjects and Nevoid Basal Cell Carcinoma Syndrome (NBCCS) patients. [Poster]
Pastorino, L.; Bertazzoni, G.; Monari, Emanuela; Cuoghi, A.; Bergamini, Stefania; Bellei, E.; Ruini, C.; Ghiorzo, P.; Bianchi Scarrà, G.; Pellacani, Giovanni; Loschi, P.; Pollio, A.; Tomasi, Aldo; Farnetani, F.; Ponti, Giovanni
abstract

PTCH1 mutations lead to complex syndromes such as the Gorlin Syndrome (GS) also named Nevoid basal Cell Carcinoma Syndrome (NBCCS, OMIM #109400) is a rare autosomal dominant disorder characterized by striking predisposition to the development of multiple basal cell carcinomas (BCCs), keratocystic odontogenic tumors (KOCTs) of the jaws, palmar and/or plantar pits and developmental defects. A variety of other benign or malignant tumors i.e., ovarian fibroma, medulloblastoma, rhabdomyosarcoma, cardiac fibroma and ameloblastoma can be found. The mechanisms underlying the increased predisposition to the development of BCCs in the context of Gorlin-Goltz syndrome is linked to molecular pathways that differ from sporadic cases. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age: moreover, the tumors typically arise on non-sunexposed skin. Fibroblasts of patients with Gorlin Syndrome may display properties determining BCC development. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and non-mutated fibroblasts. Proteomic analyses was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. The protein profiles were obtained using Copper pre-activated IMAC30 ProteinChip array. 12 protein cluster peaks, >5 kDa, had statistically significant differences in their peak intensities between PTCH1+ and PTCH1- subject groups (p<0.05). Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense vs nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies, and to develop novel experimental molecular tailored therapies based on these druggable targets. These protein peaks profiles provided better understanding of the complex skin cancer microenvironment and could be useful to select patients at risk to develop multiple and aggressive BCCs and/or other NBCCS-associated malignancies.

2013 - Proteomic profile of retained proteins from hfr cartridge [Abstract in Rivista]
Caiazzo, Marialuisa; Cuoghi, Aurora; Monari, Emanuela; Bellei, Elisa; Bergamini, Stefania; Palladino, Giuseppe; Atti, Mauro; Bruni, Francesco; Tomasi, Aldo
abstract

Hemodiafiltration with on-line endogenous reinfusion (HFR) is a dialytic method, which combines the processes of diffusion, convection and adsorption. The performance of this system is linked to the optimal combination of the membrane permeability and cartridge resin bed. Lupus nephritis (LN) remains one of the most severe manifestations of systemic lupus erythematous (LES), associated with considerable morbidity and mortality. In this preliminary study, ESI-QTOF Mass Spectrometer was used for identification of protein ultrafiltrate (UF) and for the protein captured by resin bed, obtained from one dialysed patient with LN. Methods: Plasma and UF (pre and post cartdrige) samples of one patient with LN treated with HFR, were collected at 15 min and at 235 min of the dialytic session. The cartridge utilized during treatment, containing styrenic resin, was then opened and the proteins kept by the resin were eluted by incubation O/N with 60% ACN and 1%TFA. Samples were desalted and separated by SDS-page, interesting bands were picked and “in-gel” tryptic digested before ESI-QTOF mass spectrometer analysis. Results: ESI-QTOF results of the retained proteins allowed to identifies several biomarker of kidney injury in LN, such as Retinol binding protein 4, Neutrophil gelatinase-associated lipocain, and Cystatin-C (and also TRFE, A1AG1, PTGDS, TTHY). Moreover we identify several fragments of Immunoglobulin, which are implicated in the etiopathogenesis of LES. Conclusion: The results of this preliminary study demonstrate that styrenic resin retain several proteins implicated in the LN pathogenesis, in fact the corresponding bands in the UF precartdrige at 240 min disappear confirming the removal of this proteins from the cartridge.

2013 - Superhighflux therapies for hemodialysis: ultrafiltrate proteomic profile and protein identification by on-chip elution. [Abstract in Rivista]
Monari, Emanuela; Cuoghi, Aurora; Caiazzo, Marialuisa; Bellei, Elisa; Bergamini, Stefania; Palladino, Giuseppe; Ozben, Tomris; Tomasi, Aldo
abstract

between serum levels of symmetric dimethylarginine (SDMA) and N-terminal natriuretic propeptide B-type (NT-proBNP ) and the degree of estimated glomerular filtration rate (eGFR). Methods: This study involved 98 chronic kidney disease (CKD) patients and 44 renal transplant (RT) recipients. Measurement of SDMA was determined using ELISA (DLD Diagnostica GMBH) and NT-proBNP was determined with ELFA (bioMerieux). GFR was calculated according to the Modified Diet in Renal Disease (MDRD) equation. Results: To evaluate the changes of examined parameters according to renal function we stratified CKD and RT patients to GFR categories. All patients have moderate (GFR 30–59 ml/min/1.73 m2) to severe (GFR <30 mL/min/1.73 m2) impaired renal function. Both NT-proBNP and SDMA were significantly higher in CKD and RT patients with severe than moderate impaired renal function. In CKD group, NT-proBNP values rose from 88,09 ng/L in patients with moderate eGFR to 988,78 pg/L in patients with severe eGFR (P <0.001) and from 197,05 ng/L to 996,87 ng/L in RT group (P=0.003). Obtained values for SDMA between moderate and severe eGFR were 1,09 μmol/L vs 1,62 μmol/L in CKD group and 0,58 μmol/L vs 1,18 μmol/L in RT group (P <0.001). Conclusion: NT-proBNP and SDMA are usefull for detection of severity of renal disfunction in CKD patients and renal transplant recipients.

2013 - The influence of inflammation in the search of discriminatory biomarkers for prostate cancer: a proteomic study [Abstract in Rivista]
Bergamini, Stefania; REGGIANI BONETTI, Luca; Monari, Emanuela; Bellei, Elisa; Cuoghi, Aurora; Majorana, Antonino; Ozben, Tomis; Micali, Salvatore; Sighinolfi, Maria Chiara; Tomasi, Aldo; Bianchi, Giampaolo
abstract

background: Despite the improvements in clinical and surgical practice, prostate cancer (PCa) remains one of the most widespread cancer in male. The serum marker currently used for the diagnosis of PCa is the prostate-specific antigen (PSA), but its increase does not discriminate benign prostatic hyperplasia (BPH) from PCa. In our study, we investigated the serum protein expression of BPH compared to PCa, in order to identify by Surface Enhanced Laser Desorption/Ionization - Time of Flight - Mass Spectrometry (SELDI-ToF-MS) analysis distinctive protein profiles able to unquestionably discriminate patients with a benign prostate condition from those with a malignant situation. Moreover, we considered these conditions focusing on the co-existence of inflammation. Methods: Patients with clinical suspect of PCa (PSA elevation and/or palpable mass at digital rectal exploration) and candidates for trans-rectal ultrasound guided prostate biopsy were enrolled. The analysis of protein profile of 30 patients with PCa and 30 subjects with BPH was carried out. All histological specimens were examined in order to graduate and classify the tumor and to recognize the BPH condition and presence of inflammation, that was classed in chronic and acute and then graduated in mild, moderate and severe. Serum was depleted of the 6 high-abundance proteins by immunoaffinity chromatography prior to SELDI-ToF-MS analysis. Results: The comparison between protein spectra from PCa and BPH considering the inflammation parameter and excluding samples with moderate and/or severe inflammation, identified 17 differentially expressed protein peaks using H50 ProteinChip Array.The analysis of protein profile in presence of inflammation showed different protein peaks in the two groups, some of which overlapped with those found also in the comparison between PCa and BPH in absence of inflammation. Conclusions: The inflammation seems to lead a crucial contribution in the protein profile assessments of these conditions. On the basis of our results, we believe that certain different protein peaks could be reasonably associated to inflammation rather than to cancer. Therefore, inflammation might be a confounding parameter in the search of specific biomarkers to discriminate PCa from BPH.

2013 - The omics in migraine [Articolo su rivista]
Lionetto, Luana; Gentile, Giovanna; Bellei, Elisa; Capi, Matilde; Sabato, Donata; Marsibilio, Francesco; Simmaco, Maurizio; Pini, Luigi Alberto; Martelletti, Paolo
abstract

The term omics consist of three main areas of molecular biology, such as genomics, proteomics and metabolomics. The omics synergism recognise migraine as an ideal study model, due to its multifactorial nature. In this review, the plainly research data featuring in this complex network are reported and analyzed, as single or multiple factor in pathophysiology of migraine. The future of migraine biomolecular research shall be focused on networking among these different and hierarchical disciplines. We have to look for its Ariadne's tread, in order to see the whole painting of migraine molecular biology.

2013 - Urine proteome in dogs affected by Leishmaniasis and Cushing's Syndrome [Abstract in Rivista]
Ferlizza, E; Buono, A; Fracassi, F; Dondi, F; Cuoghi, A; Bellei, E; Barrera, R; Borsetti, F; Carpenè, E; Isani, G
abstract

Urinary proteome is a topic not deeply investigated in dogs. Leishmaniasis and Cushing's Syndrome are frequently associated with persistent proteinuria. Evaluation of urine proteome with sensitive methods is considered a non invasive procedure to monitor the therapy and select novel biomarkers.

2012 - Evaluation of a possible relationship between medication-overuse headache and potential renal dysfunctions by a proteomic study on urine samples [Abstract in Rivista]
Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; Cuoghi, Aurora; Tomasi, Aldo; Guerzoni, Simona; A., Bazzocchi; Pini, Luigi Alberto
abstract

This proteomic study confirms the previous finding of alterations in urinary proteins excreted in MOH patients. Some of these proteins, identified as over-expressed particularly in NSAIDs abusers, were related to different renal dysfunctions and, probably in the development of CA. Proteomic analysis of urine proteins by the combination of 2-DE and MS could improve the knowledge of the pathophysiology of the MOH condition and identify early biomarkers to prevent the potential drug overuse-induced nephrotoxicity

2012 - Proteomic analysis of urine in medication-overuse headache patients: possible relation with renal damages [Articolo su rivista]
Bellei, Elisa; Cuoghi, Aurora; Monari, Emanuela; Bergamini, Stefania; Fantoni, Luca Isaia; Zappaterra, Maurizio; Guerzoni, Simona; Bazzocchi, Annalisa; Tomasi, Aldo; Pini, Luigi Alberto
abstract

Medication-overuse headache (MOH) is a chronic disorder associated with overuse of analgesic drugs, triptans, non-steroidal anti-inflammatory drugs (NSAIDs) or other acute headache compounds. Various epidemiologic investigations proved that different drug types could cause nephrotoxicity, particularly in chronic patients. The aim of the present work was to analyze, by aproteomic approach, the urinary protein profiles of MOH patients focusing on daily use of NSAIDs, mixtures and triptans that could reasonably be related to potential renal damage. We selected 43 MOH patients overusing triptans (n = 18), NSAIDs (n = 11), and mixtures (n = 14), for 2–30 years with a mean daily analgesic intake of 1.5 ± 0.9 doses, and a control group composed of 16 healthy volunteers. Urine proteins were analyzed by monodimensional gel electrophoresis and identified by mass spectrometry analysis. Comparing the proteomic profiles of patients and controls, we found a significantly different protein expression, especially in the NSAIDs group, in which seven proteins resulted over-secreted from kidney (OR = 49, 95% CI 2.53–948.67 vs. controls; OR = 11.6, 95% CI 0.92–147.57 vs. triptans and mixtures groups). Six of these proteins (uromodulin, a-1-microglobulin, zinc-a-2- glycoprotein, cystatin C, Ig-kappa-chain, and inter-a-trypsin heavy chain H4) were strongly correlated with various forms of kidney disorders. Otherwise, in mixtures and in triptans abusers, only three proteins were potentially associated to pathological conditions (OR = 4.2, 95% CI 0.33–53.12, vs. controls). In conclusion, this preliminary proteomic study allowed us to define the urinary protein pattern of MOH patients that is related to the abused drug. According with the obtained results, we believe that the risk of nephrotoxicity should be considered particularly in MOH patients who abuse of NSAIDs.

2012 - Proteomic profile of non-bacterial proteins in periodontal pockets: a pilot study. [Abstract in Rivista]
Simonazzi, L.; Monari, Emanuela; Ferrari, Davide; Bellei, Elisa; Melpignano, F.; Cuoghi, Aurora; Bergamini, Stefania; Pellacani, C.; Campedelli, F.; Tomasi, Aldo; Bertoldi, Carlo
abstract

Aim: In this study, we attempted to identify the proteins involved in periodontitis comparing the proteomic profile of interproximal pocket tissue affected by the periodontal lesion with interproximal healthy tissue in the same subject, in sites where no periodontopathogenic bacteria were detectable. Material and Methods: Using specific inclusion and exclusion criteria, we enrolled 15 subjects affected by severe chronic periodontitis. The subjects presenting at least one intrabony defect (next to a healthy interproximal site to be included in the flap design) suitable for treatment by osseous respective surgery, were considered eligible for this study. Biopsies of connective tissue were harvested from the intrabony component of the defect and from healthy tissue (from the secondary flap) and immediately frozen at –80°C. A two-dimensional gel electrophoresis system was used to detect differences in protein expression between pathologic and healthy tissue and protein identification by LC-MS/MS analysis was performed. Results: Six proteins (tropomyosin α-4 chain, 14-3-3 protein z/δ, putative heat shock protein HSP 90-β, peroxiredoxin-1, fatty acid binding protein, S100-A9) displayed a higher while five a lower (annexin A1, actin cytoplasmatic-1, carbonic anhydrase-2, Ig K chain C region and flavin reductase) protein expression level in pathologic tissue compared with healthy tissue. Conclusion: The highlight of proteins involved in the immunological and cytokine signaling cascade mediation or in connective and epithelial regeneration and differentiation shows that the pathogenic process is active in periodontal sites also in absence of periodontopathogen microbiota. Besides, proteomic analysis could be considered very useful in studying the pathogenesis of periodontitis. Abstract N: P0224 ISSN:1600-051X (Electronic).

2012 - Quantification of P-cresol sulphate in human plasma of uremic patients by MRM [Abstract in Rivista]
Cuoghi, Aurora; Bellei, Elisa; Caiazzo, Marialuisa; Bergamini, Stefania; G., Palladino; Monari, Emanuela; Tomasi, Aldo
abstract

n/a

2012 - Quantification of p-cresol sulphate in human plasma by selected reaction monitoring. [Articolo su rivista]
Cuoghi, Aurora; M., Caiazzo; Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; G., Palladino; T., Ozben; Tomasi, Aldo
abstract

Chronic renal failure patients accumulate in the blood molecules that are normally excreted into the urine. p-Cresol Sulphate (pCS), the most representative retained toxin, shows a high level of toxicity. Therefore, its quantification could represent a prediction factor to determine the risk of endothelial dysfunction and cardiovascular complication and response to the haemodialysis treatment. The aim of this study was to evaluate the suitability of the multiple reaction monitoring (MRM) technique in order to improve the sensibility, the selectivity and the timing of pCS detection in a small amount of plasma. Deproteinized plasma of uremic patients was concentrated and dissolved in liquid chromatography (LC) mobile phase solution. pCS was quantified by LC coupled to tandem mass spectrometry (LC-MS/MS) on a triple-quadrupole mass spectrometer. Selective and sensitive detection of pCS was achieved by selecting the specific parent ion and monitoring two specific fragment ions. The MRM assay was carried out using the following transitions: m/z 187 → 80.00 and m/z 187 → 107.00. A good linearity was observed for each calibration curve. The intra-day and inter-day results showed a good precision and repeatability. The percentage recoveries indicate an optimal selectivity of the analytical method. The MRM assay to quantify pCS in a small amount of human plasma is rapid, highly sensitive, selective and with a good repeatability.

2012 - Quantificazione del p-cresol solfato nel plasma di pazienti uremici mediante MNR. [Abstract in Atti di Convegno]
Cuoghi, Aurora; Bellei, Elisa; Caiazzo, Marialuisa; Bergamini, Stefania; G., Palladino; Tomasi, Aldo; Monari, Emanuela
abstract

n/a

2012 - Steps Study: Superhighflux therapies for hemodialysis. A proteomic approach. [Abstract in Rivista]
Caiazzo, Marialuisa; Monari, Emanuela; Cuoghi, Aurora; Bellei, Elisa; Bergamini, Stefania; G., Palladino; Tomasi, Aldo
abstract

n/a

2012 - Studio STEPS: Superior Therapies for hemodialysis. Approccio proteomico. [Abstract in Atti di Convegno]
Caiazzo, Marialuisa; Cuoghi, Aurora; Monari, Emanuela; Bellei, Elisa; Bergamini, Stefania; G., Palladino; Tomasi, Aldo
abstract

n/a

2011 - Can prostatitis to be a confounding parameter in prostatic proteomic profile designation? [Abstract in Rivista]
Bergamini, Stefania; REGGIANI BONETTI, Luca; Monari, Emanuela; Bellei, Elisa; Maiorana, Antonino; Ozben, T.; Tomasi, Aldo; Micali, Salvatore; Bianchi, Giampaolo
abstract

N/A

2011 - Effect of nebivolol treatment on proteomic profiling during atherosclerosis progression. [Abstract in Atti di Convegno]
Ozben, T.; Ozben, B.; Dursun, E.; Monari, Emanuela; Cuoghi, Aurora; Bellei, Elisa; Bergamini, Stefania; Tomasi, Aldo
abstract

na

2011 - Enriched sera protein profiling for detection of non-small cell lung cancer biomarkers. [Articolo su rivista]
Monari, Emanuela; Casali, Christian; Cuoghi, Aurora; Nesci, Jessica; Bellei, Elisa; Bergamini, Stefania; Fantoni, Luca Isaia; Natali, Pamela; Morandi, Uliano; Tomasi, Aldo
abstract

BackgroundNon Small Cell Lung Cancer (NSCLC) is the major cause of cancer related-death. Many patients receive diagnosis at advanced stage leading to a poor prognosis. At present, no satisfactory screening tests are available in clinical practice and the discovery and validation of new biomarkers is mandatory. Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) is a recent high-throughput technique used to detect new tumour markers. In this study we performed SELDI-ToF-MS analysis on serum samples treated with the ProteoMinerTM kit, a combinatorial library of hexapeptide ligands coupled to beads, to reduce the wide dynamic range of protein concentration in the sample. Serum from 44 NSCLC patients and 19 healthy controls were analyzed with IMAC30-Cu and H50 ProteinChip Arrays.ResultsComparing SELDI-ToF-MS protein profiles of NSCLC patients and healthy controls, 28 protein peaks were found significantly different (p<0.05), and were used as predictors to build decision classification trees. This statistical analysis selected 10 protein peaks in the low-mass range (2-24 kDa) and 6 in the high-mass range (40-80 kDa). The classification models for the low-mass range had a sensitivity and specificity of 70.45% (31/44) and 68.42% (13/19) for IMAC30-Cu, and 72.73% (32/44) and 73.68% (14/19) for H50 ProteinChip Arrays.ConclusionsThese preliminary results suggest that SELDI-ToF-MS protein profiling of serum samples pretreated with ProteoMinerTM can improve the discovery of protein peaks differentially expressed from NSCLC patients and healthy subjects, useful to build classification algorithms with high sensitivity and specificity. However, identification of the significantly different protein peaks needs further study in order to provide a better understanding of the biological nature of these potential biomarkers and their role in the underlying disease process.

2011 - High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins. [Articolo su rivista]
Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Fantoni, Luca Isaia; Cuoghi, Aurora; Ozben, T; Tomasi, Aldo
abstract

In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.

2011 - Neutralization by human serum samples of a transmissible agent isolated from the cerebrospinal fluid of neurological patients. [Articolo su rivista]
Beretti, Francesca; P., Pietrosemoli; A. M., Bartoletti; M., Gerolmini; Bellei, Elisa; Tomasi, Aldo; Portolani, Marinella
abstract

A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in severalcerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusualinfectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such anactivity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showeda blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection,two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). Whenneutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein(s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samplesdisclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reportedin previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymesMn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathwayamong the causes of the DNA damage induced by the cytotoxic transmissible agent under study.

2011 - Optimizing protein recovery yield from serum samples treated with beads technology. [Articolo su rivista]
Bellei, Elisa; Monari, Emanuela; Bergamini, Stefania; Ozben, T.; Tomasi, Aldo
abstract

Proteomics studies are often complicated by the wide dynamic range of the biologicalfluids, in which few highly abundant proteins obscure the signal of low abundant ones.To overcome this problem, several techniques have been developed on the basis of‘‘depletion principles,’’ namely immuno-subtraction with specific antibodies against themost-abundant proteins. Unfortunately, the probability of codepletion is a noteworthydrawback associated with these strategies. The ProteoMinerTM (PM) technology is anovel approach, consisting of a combinatorial library of hexapeptide ligands coupled tobeads, that allows the capture of all species present in a proteome, but at much reducedprotein concentration differences, simultaneously enhancing the concentration of themost dilute species. In this study, we evaluated the compatibility of the PM kit’s elutionreagent with 2-DE analysis, comparing five different purification methods on serumsamples eluted from the beads: the ‘‘ReadyPrep 2-D Clean-up kit’’ and precipitation withorganic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol.Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitationwith ACN offered the most promising approach, showing the best spot resolution inall regions of the pH gradient and the greatest number of protein spots visualized on 2-Dgels.

2011 - Proteomic identification of periodontal pocket involved proteins: a pilot study. [Abstract in Rivista]
Bertoldi, Carlo; Bellei, Elisa; Cuoghi, Aurora; Pellacani, C.; Tomasi, Aldo; Monari, Emanuela
abstract

Vedi allegato

2011 - Urine proteomic analysis in patients with cronic headache and overuse of analgesic drugs: possible relation with renal damage [Abstract in Rivista]
Bellei, Elisa; Bergamini, Stefania; Cuoghi, Aurora; Monari, Emanuela; Tomasi, Aldo; Zappaterra, Maurizio; Bazzocchi, Annalisa; Guerzoni, Simona; Pini, Luigi Alberto
abstract

NA

2010 - POTENTIAL ROLE OF PROTEOMIC ANALYSIS IN PROSTATE CANCER DIAGNOSIS [Abstract in Rivista]
Bianchi, Giampaolo; Sighinolfi, Maria Chiara; Bergamini, Stefania; Bellei, Elisa; Monari, Emanuela; Cuoghi, Aurora; Micali, Salvatore; DE STEFANI, Stefano; Tomasi, Aldo
abstract

not available

2009 - Analisi proteo mica nella neoplasia prostatica: Risultati preliminari e nuove prospettive. [Poster]
Bianchi, Giampaolo; Sighinolfi, M. C.; Micali, Salvatore; DE STEFANI, Stefano; Bergamini, Stefania; Bellei, E.; Monari, Emanuela; Cuoghi, A.; Tomasi, Aldo
abstract

Analisi proteo mica nella neoplasia prostatica: Risultati preliminari e nuove prospettive.

2009 - Biomarkers discovery of renal alterations in urine of diabetic patients by proteomic analysis [Abstract in Rivista]
Tomasi, A; Bellei, E; Bergamini, S; Monari, E; Cuoghi, A; Albertazzi, A; Lucchi, L
abstract

Discovery of potential biomarkers correlated to renal alterations in urine of diabetic patients by proteomic analysis.

2009 - Free radical induced oxidative protein modification in kidney diseases: proteomic detection [Abstract in Atti di Convegno]
Bergamini, S; Bellei, E; Monari, E; Cuoghi, A; Fantoni, Li; Lucchi, L; Tomasi, A
abstract

Proteomic detection of free radical induced oxidative protein modification in kidney diseases

2009 - PROTEOMICS AS DIAGNOSTIC MARKERS [Abstract in Rivista]
Tomasi, Aldo; Bellei, Elisa; Bergamini, Stefania; Cuoghi, Aurora; Monari, Emanuela
abstract

non disponibile

2009 - Proteomic analysis to discover early urinary biomarkers of type 2 diabetic nephropathy [Poster]
Bellei, E; Cuoghi, A; Monari, E; Lucchi, L; Bergamini, S; Tomasi, A
abstract

Objectives. Nephropathy associated with type 2 diabetes mellitus (T2D) is the most frequent cause of end stage renal disease (ESRD) in all Western countries, and a major healthcare problem. In the United States, the incidence of diabetic nephropathy (DN) has increased by 150% in the past 10 years: a similar trend has been reported also in Europe. Despite rapid research progress, predictors able to assess prospectively with high precision the risk for DN in individuals with diabetes are still lacking. Thus, at present the available therapies are usually initiated at more advanced stages of DN, characterized by evident clinical manifestations and progressive decline in glomerular filtration rate (GFR), leading toward both ESRD and cardiovascular events. In this study we made a comparison between the proteomic pattern of a group of normoalbuminuric T2D patients with those of a group of T2D patients with DN (T2DN). Both were compared with a group of healthy subjects, in the search of a diagnostic proteomic pattern that could help to identify patients at increased risk of kidney disease progression. Methods. We used two different proteomic approaches: the Surface-Enhanced Laser Desorption Ionization-Time of Flight technique (SELDI-TOF) and the two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MS). We performed urinary proteomic analysis of 10 T2D patients with microalbuminuria in the normal range, 12 T2DN patients, and 12 healthy subjects as normal controls. For protein profiling with SELDI-TOF, urine samples were analyzed using immobilized metal affinity capture (IMAC30), weak cation exchanger (CM10) and reverse phase (H50) ProteinChip Arrays. Statistical analysis was performed in the obtained spectra (Ciphergen Express 3.0) in order to identify peaks that showed significant differences among groups. Furthermore, urinary proteins were separated and visualized by 2-DE; the differentials spot were selected and identified by MS, using an ESI-Q-TOF mass spectrometer. Results. Comparing the patients proteomic profiles with those of normal controls, we identified 11 progressively and differently expressed proteins. The majority of these proteins resulted significantly increased in both patients groups, and corresponded to: transthyretin precursor, plasma retinol-binding protein, -2-microglobulin, carbonic anhydrase, -2-glycoprotein 1 (or apolipoprotein H), Ig kappa chain C region, Ig kappa chain V-III region SIE and Ig kappa chain V-II region Cum (or Bence-Jones protein). Only 3 proteins were gradually decreased in patients groups, including the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein-3. Conclusions. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN, but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage and could help to identify the diabetic patients prone to progress toward DN.

2009 - Proteomics to identify free radical induced oxidative protein modifications as biomarkers associated to type 2 diabetic nephropathy [Abstract in Atti di Convegno]
Bellei, E; Bergamini, S; Lucchi, L; Monari, E; Cuoghi, A; Fantoni, Li; Tomasi, A
abstract

Proteomics as a tool to identify free radical induced oxidative protein modifications as candidate biomarkers associated to type 2 diabetic nephropathy

2008 - Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients [Articolo su rivista]
Bellei, Elisa; Rossi, Elena; Lucchi, L; Uggeri, S; Albertazzi, Alberto; Tomasi, Aldo; Iannone, Anna
abstract

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic add phosphatase precursor, the ribonuclease and the kallikrem-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig K chain C region, Ig K chain V-II region Cum, Ig K-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.

2007 - Detection of type 2 diabetic nephropathy-associated biomarkers using proteomic tachniques [Abstract in Rivista]
Tomasi, Aldo; Bellei, Elisa; Albertazzi, Alberto; Monari, Emanuela; Lucchi, L.
abstract

not available

2007 - Urinary protein profiling for biomarker discovery in diabetic nephropathy [Abstract in Rivista]
Tomasi, Aldo; Bellei, Elisa; Monari, Emanuela; Albertazzi, Alberto; Lucchi, L.
abstract

Renal disease in patients with Type II diabetes is the leading cause of terminal renal failure and a major healthcare problem. Diabetic renal damage might be reflected by a change in urinary proteins and polypeptides excretion at a very early stage. The characterization of urinary polypeptides is of great clinical interest and significance, yielding to a better understanding of the changes within the kidney during the development of diabetic nephropathy (DN).Proteomics has now become an emerging field in the post-genomic area and offers the opportunity of large-scale protein analysis. Two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) combined with mass spectrometry (MS) is commonly used for protein separation and to determine the molecular weight of polypeptides and proteins. Surface-Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS), which represents the succesful combination of retentate chromatography and MS, is an alternative technology employed for high-throughput analysis of biological samples.The aim of this study was to identify urinary protein patterns in Type II diabetic subjects, that may serve as diagnostic tool for the early recognition and development of DN.In our study, we included 25 Type II diabetes mellitus patients with no nephropathy, 25 Type II diabetes mellitus patients with nephropathy (identified by serum creatinine level < 2mg/dl and microalbuminuria) and 20 healthy volunteers. Morning midstream urine samples were collected, centrifuged to remove cell debris and precipitated with acetone/methanol at -20°C. After centrifugation, the protein pellet was resuspended in a specific solubilization buffer and finally 100 micrograms of protein were subjected to 2DE-PAGE. Silver-stained gels were analyzed by the PDQuest® Image Analysis software, differential proteins were cut and identified by LC-ESI-MS/MS.For protein profiling with SELDI-TOF-MS, urine samples were analyzed using immobilized metal affinity capture (IMAC30), strong anionic exchanger (Q10), weak cation exchanger (CM10), reverse phase (H50) ProteinChip® Arrays and sinapinic acid (SPA) as matrix, according to the manufacturer’s instructions (Ciphergen, USA). Urinary protein profiles were collected in the range 0-200.000 m/z, using different acquisition protocols and different laser energies. Statistical analysis was performed in the obtained spectra (Ciphergen Express 3.0) in order to identify peaks that showed significant differences among groups.Two characteristics proteins (prostaglandin-H2 D-isomerase and antithrombin-III) were identified in the urine of Type II diabetes patients compared to healthy individuals using 2DE-PAGE coupled to MS. In addition to these two proteins, the following proteins were identified in urine of patients with Type II diabetes mellitus with nephropathy: vitamin-D binding protein, gelsolin, apolipoprotein A-IV, plasma retinol binding protein and lipocalin-1, with an increased amount of albumin.In conclusion, comparative data analysis discovered distinct proteins characteristic for diabetic patients and leads to the establishment of possible individual risk factors for the development of DN, which may contribute to clinical staging in the future. These techniques may also enable the identification of novel proteins related to the pathophysiology of DN, which may serve as therapeutical targets

2006 - 2-D proteomic analysis of urine in diabetic patients with nephropathy [Abstract in Rivista]
Tomasi, Aldo; L., Lucchi; Uggeri, Simona; Bellei, Elisa; Rossi, Elena; Albertazzi, Alberto; Iannone, Anna
abstract

Mortality and morbidity associated with diabetes is diabetic nephropathy. We carried out a research project on the identification of specific proteomic profiles in patients with diabetic nephropathy. The rationale for this study is based on studies demonstrating that chronic inflammation and oxidative stress determine the complications observed in diabetic patients. Such a pathogenesis will determine post-translational protein modification, bringing to the identification of prognostic and predictive markers. Recent advances in proteomic profiling technologies have allowed preliminary profiling. Various specific proteomic profiles have been described for various pathologies, in particular tumors. Studies on proteomic profiling in diabetic nephropathy have not been performed yet. We analyzed easily available biological samples such as urine to evaluate the expression and function levels of urinary molecules that could become diagnostic, prognostic and predictive markers of disease.

2006 - Caratterizzazione di proteine nel tessuto e nel liquido interstiziale di neoplasie della mammella [Abstract in Atti di Convegno]
Barchetti, A; Cortesi, L; Rossi, E; Bellei, E; Tomasi, A; Iannone, A; Federico, M.
abstract

La classificazione dei tumori della mammella si basa attualmente sui parametri clinici e sulla morfologia cellulare; l’analisi immunoistochimica utilizza un numero ristretto di fattori prognostici e predittivi correlati a diverse funzioni cellulari come la proliferazione, l’angiogenesi, l’invasione e la metastatizzazione. Questi parametri si limitano a classificare i pazienti in sottogruppi con differente prognosi mentre l’uso di nuove tecnologie, quali lo studio delle proteine attraverso elettroforesi bidimensionale (2D), potrebbe portare ad un approccio terapeutico individualizzato e ad una ottimizzazione del trattamento. Lo scopo del nostro studio è quello di individuare nuovi markers nell’ambito dei tumori invasivi della mammella confrontando il profilo di espressione proteica di tessuto normale, patologico, di TIF (tumoral interstitial fluid) e di NIF (normal interstitial fluid). Nel microambiente tumorale si concentrano infatti un gran numero di molecole in parte secrete, in parte trasportate da vescicole di membrana o liberate in seguito a morte cellulare. Abbiamo ottenuto 8 biopsie mammarie da pazienti affette da carcinoma duttale infiltrante (G3) e carcinoma lobulare infiltrante (G2/3). L’indagine è stata condotta utilizzando circa 200 mg di tessuto mammario di carcinoma duttale e di tessuto adiacente sano. I frammenti di biopsia, sospesi in tampone PBS, sono stati incubati in CO2 5% a 37°C per un’ora e successivamente centrifugati 20 min. a 3000g. Da questa coltura è stato recuperato e analizzato il surnatante contenente le proteine rilasciate dal tessuto. Successivamente l’estrazione proteica è stata effettuata anche sul pellet di tessuto bioptico polverizzato in azoto e sonicato in tampone di lisi. Per ciascuna analisi 100ug di proteine sono state sottoposte ad elettroforesi 2D e visualizzate con metodica silver staining. Gli spots di interesse sono stati caratterizzati con la tecnica HPLC-ESI-MS/MS in seguito a purificazione e digestione con tripsina. L’analisi preliminare delle mappe bidimensionali attraverso il programma PDQuest (BIO-RAD) ha evidenziato spots proteici la cui intensità variava significativamente nei campioni tumorali. Da segnalare un’aumentata espressione dell’oncogene mitogeno DJ-1(PARK 7), coinvolto in pathways di trasduzione del segnale Ras dipendenti e delle proteine LYPA (Acyl-protein thioesterase) e LDHB (lactate dehydrogenase B) la cui overespressione è stata segnalata in linee cellulari di tumore mammario resistenti al trattamento anti-estrogenico. È stato inoltre riscontrato un aumentato livello di PSA1 (Prostatic Specific Antigen), una proteasi serinica associata in letteratura ad un basso indice di proliferazione e ad una prognosi favorevole. L’analisi e il confronto di un maggiore numero di casi permetterà di chiarire il significato e di ottenere informazioni specifiche sul pattern di espressione genica dei differenti stadi e istotipi del carcinoma mammario. Inoltre il confronto con pattern proteici sierici potrebbe essere di aiuto nell’identificazione di nuovi e predittivi marker ematici di neoplasia.

2006 - Markers urinari di nefropatia diabetica [Poster]
Bellei, E; Rossi, E; Lucchi, L; Uggeri, S; Albertazzi, A; Tomasi, A; Iannone, A
abstract

Il diabete, come riportato ampiamente in letteratura, è una delle principali cause di insufficienza renale in stadio terminale (ESRD). Nonostante i progressi raggiunti nella comprensione della nefropatia diabetica, i meccanismi patogenetici che inducono il danno non sono ancora ben definiti. Alcuni studi hanno evidenziato come l’infiammazione cronica e i danni ossidativi ad essa correlati contribuiscano in modo determinante alle complicanze osservate in questi pazienti. Si può inoltre ipotizzare la presenza di modificazioni post-traduzionali di alcune proteine, che potrebbero essere impiegate come biomarkers prognostici e predittivi di malattia. Profili proteomici altamente specifici sono stati caratterizzati per varie patologie, soprattutto tumorali, ma indagini su profili proteomici nella nefropatia diabetica umana non sono tuttora presenti in letteratura. In questo studio abbiamo confrontato pattern proteici di soggetti sani con quelli di pazienti con nefropatia diabetica, cercando di individuare quadri proteici caratteristici dei diversi stadi di patogenesi, nonché modifiche post-traduzionali peculiari o mutazioni correlate alla patologia in esame. Partendo da liquidi biologici facilmente reperibili e maneggiabili come le urine, è possibile infatti valutare, mediante elettroforesi bidimensionale (2-DE), la funzionalità e i differenti livelli di espressione di molecole urinarie. Sono stati selezionati 20 pazienti con diabete di tipo II associato a nefropatia (valori di creatininemia > 1,5 mg/dl e presenza di microalbuminuria). Alle urine, immediatamente dopo la raccolta, è stato aggiunto un cocktail di inibitori delle proteasi. Una aliquota di ciascun campione è stata precipitata in acetone a -20°C, centrifugata e il pellet risospeso in un opportuno tampone. Dopo aver misurato la concentrazione proteica totale tramite un kit commerciale, 10 microgrammi di proteine sono stati utilizzati per l’analisi bidimensionale. I gel ottenuti sono stati colorati con metodica Silver stain massa-compatibile, gli spots di interesse trattati con tripsina e i peptidi estratti identificati tramite spettrometria ESI-LC- MS/MS. I risultati preliminari hanno evidenziato un aumento dei livelli delle seguenti proteine nelle urine dei pazienti diabetici nefropatici: albumina, transferrina e immunoglobuline G (IgG), che dalla letteratura risultano essere indici di danno glomerulare; alpha-1-microglobulina e alpha-1-antitripsina, indicatori di alterata funzionalità tubulare. Inoltre altre tre glicoproteine (zinc-alpha-2-glicoprotein, alpha-1-acid-glicoprotein e leucine-rich-alpha-2-glicoprotein), risultano quantitativamente aumentate. La presenza invece di altri due indicatori di danno tubulare, ovvero beta-2-microglobulina (b-2MG) e retinol binding protein (RBP), oltre alla proteina di membrana perlecan, è stata riscontrata solamente nelle urine dei pazienti e non in quelle dei soggetti di controllo. In conclusione, la 2-DE accoppiata alla spettrometria di massa potrebbe rappresentare un metodo rapido, sensibile e non-invasivo per identificare markers precoci di patologia renale.

2006 - Ricerca di biomarkers nel tumore ovarico mediante analisi proteomica [Abstract in Atti di Convegno]
Rossi, E; Cortesi, L; Barchetti, A; Bellei, E; Monari, E; Tomasi, A; Iannone, A; Abrate, M; Federico, M.
abstract

Il carcinoma ovarico rappresenta la quarta causa di morte nel sesso femminile in Europa e negli Stati Uniti. Nell’80 % dei casi infatti, la diagnosi avviene in uno stadio avanzato poiché la maggior parte delle neoplasie ovariche rimane clinicamente asintomatica negli stadi I e II. La sola terapia chirurgica assicura una sopravvivenza a cinque anni del 90 % delle pazienti diagnosticate allo stadio I, percentuale che si riduce al 35% negli stadi più avanzati. Il nostro obbiettivo è quello di utilizzare l’elettroforesi bidimensionale (2DE) e la spettrometria di massa per identificare e caratterizzare nuovi specifici biomarkers tumorali ovarici che permettano di effettuare una diagnosi precoce, di monitorare la progressione tumorale e di impostare una terapia mirata. Le indagini preliminari sono state effettuate su campioni bioptici di tessuto ovarico sano e tumorale, e su liquido proveniente da lavaggio peritoneale. Le proteine sono state estratte da circa 100 mg di tessuto congelato in azoto liquido, polverizzato in un mortaio e risospeso in tampone di lisi contenente urea, tiourea, CHAPS, Tris-HCl, ampholine pH 3-10 e inibitori delle proteasi. Dopo incubazione a temperatura ambiente per un’ora, i campioni sono stati sonicati e centrifugati. Il pellet, ottenuto dopo precipitazione in acetone freddo, è stato risospeso in un buffer di reidratazione (urea, tiourea, CHAPS, DTT e ampholine) e il contenuto proteico totale misurato con il metodo Bradford. L’isoelettrofocalizzazione (IEF) è stata effettuata caricando 80 g di proteine totali su IPG strips a range di pH 3-10 non lineare, fino al raggiungimento di 70000 Vh finali. Le proteine focalizzate sono state ridotte con DTT e alchilate con Iodoacetamide in tampone di equilibrazione (Urea, Tris-HCl, Glicerolo, SDS, Blu di Bromofenolo) prima della separazione elettroforetica su gels di poliacrilamide (SDS–PAGE), a concentrazione omogenea 10% e a gradiente 8-16%. I gel sono stati infine colorati con metodica Silver-staining massa-compatibile. L’analisi con il programma PDQuest ha evidenziato la presenza di numerosi spot proteici differenzialmente espressi nei campioni tumorali rispetto ai controlli sani; la spettrometria di massa ESI-Q-TOF-MS/MS porterà all’identificazione delle proteine di interesse. Per quanto riguarda il liquido derivato da lavaggio peritoneale, prima dell’analisi 2DE le aliquote prelevate sono state concentrate con filtri Amicon aventi cut-off di PM 5 KDa. Parallelamente alla 2DE, sui campioni tissutali è stata effettuata anche l’analisi con tecnologia SELDI-TOF (Surface Enhanced Laser Desorption Ionisation-Time of Flight). I risultati ottenuti hanno evidenziato variazioni significative dell’espressione di numerose proteine, soprattutto a basso peso molecolare (<20 KDa). Lo scopo dello studio ha la finalità di giungere all’identificazione di biomarcatori specifici del tumore ovarico che possono essere evidenziati anche nel liquido peritoneale ed eventualmente nel siero, fungendo da fattori prognostici della malattia ed anche predittivi di risposta al trattamento con derivati del platino.

2006 - Ricerca di markers diagnostici, prognostici e predittivi in carcinomi mammari attraverso analisi proteomica [Abstract in Atti di Convegno]
Pecorari, L; Silvestri, C; Candini, O; Rossi, E; Bellei, E; Bussolari, R; Iannone, A; Federico, M; Cortesi, L; Calabretta, B.
abstract

Le basi molecolari dei tumori sporadici della mammella sono solo in parte conosciute: un comune meccanismo molecolare, che è stato riportato in circa il 75% dei casi, è l’attivazione della proteina oncogenica ad attività Serin/Treonin chinasica AKT. Una delle principali funzioni di pAKT (la cui forma attiva è fosforilata) è quella di promuovere la sopravvivenza cellulare mediata da fattori di crescita bloccando i meccanismi intrinseci dell’apoptosi. Recentemente numerosi studi si sono concentrati sull’approfondimento del ruolo dell’oncoproteina AKT nel carcinoma mammario; la conclusione comune a questi studi è che si possano ottenere benefici clinici rilevanti dall’appropriata combinazione terapeutica tra chemioterapici convenzionali e inibitori specifici di pAKT. Sarebbe inoltre di notevole importanza chiarire in che modo la valutazione dell’attività di pAKT possa essere interpretata ai fini diagnostici, prognostici e predittivi in pazienti con carcinoma mammario. Questo progetto di ricerca si propone di approfondire questi aspetti utilizzando un approccio basato su tecniche di analisi proteomica. Su diversi tipi di linee continue di carcinoma mammario con diverse caratteristiche e diverso grado di aggressività (ErbB2+ / ErbB2- / BRCA1 mutato / BRCA1 w.t.) sono stati eseguiti e sono tuttora in corso, esperimenti d’inibizione/deplezione della proteina pAkt. In particolare dalla linea SKBr3 è stato possibile ottenere estratti proteici contenenti una discreta quota di pAKT; inoltre esperimenti di immunoprecipitazione selettiva hanno consentito di isolare gli interattomi di pAKT e di AKT non fosforilata. Questi immunoprecipitati sono poi stati sottoposti ad elettroforesi bidimensionale e gli spot indicativi di proteine varianti (associate quindi ad una forma rispetto all’altra) analizzati tramite spettrometria di massa. Attualmente una sola nuova potenziale proteina cliente, Elongation Factor 1 alfa (EF1a), è stata identificata ed è in via di caratterizzazione. Per approfondire il ruolo di questa proteina (nota per essere iperespressa in diversi tipi di carcinoma tra i quali l’ovarico, il mammario e il prostatico) sono stati condotti studi di “RNAinterference” che hanno permesso di asserire che in cellule di carcinoma mammario la riduzione dell’espressione di EF1a (livelli diminuiti del 60%) porta ad una riduzione del 30% della clonogenicità. Sembra inoltre che diminuiti livelli d’espressione di EF1 associati a trattamenti a base di 17AAG (inibitore selettivo dell’attività di HSP90 che causa forti riduzioni ai livelli di pAKT) abbiano un effetto sinergico nella riduzione della proliferazione cellulare. Parallelamente a questi studi, stiamo programmando un’analisi globale di tutte le proteine che manifestano una espressione differenziale dopo l’inibizione di pAKT (globale, perché non si riferisce alle sole proteine dell’interattoma, ma all’estratto totale). Questo verrà realizzato tramite confronto diretto delle mappe proteiche ottenute dopo elettroforesi bidimensionale degli estratti totali (trattamento inibitorio specifico vs. controllo) individuando e identificando gli “spots” che rappresentano proteine la cui espressione varia a seguito dell’inibizione dell’attività chinasica di pAKT. La scoperta di nuove proteine substrato della attività chinasica di pAKT e l’approfondimento dei meccanismi biologici e molecolari da essa regolati, rappresentano un importante punto di partenza per la progettazione di strategie terapeutiche ottimali e per la validazione di nuovi markers diagnostici, prognostici o predittivi in situazioni di carcinoma mammario.

2005 - Effetto della N-acetilcisteina sulla progressione dell'insufficienza renale cronica nel ratto [Poster]
Bellei, E; Bergamini, S; Ligabue, G; Ballestri, M; Albertazzi, A; Tomasi, A; Iannone, A.
abstract

L’insufficienza renale cronica porta nel tempo ad un progressivo deterioramento della funzionalità renale, sino ad arrivare a quella che viene definita come “end-stage renal disease” (ESRD). I meccanismi coinvolti in questo processo multifattoriale sono mediati dall’azione di diverse sostanze, fra cui: angiotensina II, TGF, endotelina, monossido d’azoto (NO), radicali liberi dell’ossigeno e citochine. Si è voluto indagare l’effetto di una dieta arricchita con una sostanza antiossidante e anti-infiammatoria, la NAC, sulla progressione dell’insufficienza renale cronica indotta in ratti mediante asportazione chirurgica dei 5/6 del parenchima renale.

2005 - Oxidative stress and experimental shock [Abstract in Rivista]
Tomasi, Aldo; Bergamini, Stefania; Bellei, Elisa; Bazzani, Carla; Bertolini, Alfio; Guarini, Salvatore; Iannone, Anna
abstract

In this introductory presentation, some of the recent results of our and other groups on the oxidative stress theory in the development of experimental shock, will be presented and discussed.

2005 - Oxidative stress in renal patients [Abstract in Rivista]
Iannone, Anna; Bergamini, Stefania; Bellei, Elisa; Rota, Cristina; DELLA CASA, Lara; Tomasi, Aldo
abstract

non disponibile

2004 - Antiossidanti come modulatori del metabolismo degli acidi grassi [Abstract in Atti di Convegno]
Banni, S; Melis M., P; Carta, G; Cordeddu, L; Murru, E; Bellei, E; Iannone, A
abstract

Antiossidanti come modulatori del metabolismo degli acidi grassi

2004 - Chronic inflammation in end stage renal disease: markers of oxidative stress and redox modification [Abstract in Atti di Convegno]
Iannone, A; Bergamini, S; Bellei, E; Rota, C; Tomasi, A
abstract

Markers of oxidative stress and redox modification in patients with end stage renal disease and chronic inflammation

2004 - Effect of alpha-tocopherol and N-acetylcysteine on benzoyl peroxide toxicity in human keratinocytes [Articolo su rivista]
Bellei, Elisa; Rota, Cristina; Bergamini, Stefania; P., Manfredini; Albertazzi, Alberto; Tomasi, Aldo; Iannone, Anna
abstract

Benzoyl peroxide is a free-radical generating compound widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, benzoyl peroxide causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, benzoyl peroxide induces DNA strand breaks in many cells, including keratinocytes. Benzoyl peroxide toxicity is presumably mediated by the formation of reactive free radicals and by the consumption of intracellular antioxidants. In this work we investigated the effect of both the lipophilic antioxidant et-tocopherol and the hydrophilic thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to benzoyl peroxide. A protective effect against benzoyl peroxide cytotoxicity was achieved when cells were grown on a alpha-tocopherol layer. On the contrary, the addition of alpha-tocopherol dissolved in ethanol had a pro-oxidant effect, leading to an enhancement of benzoyl peroxide toxicity. Cytotoxicity was also reduced adding NAC to the culture medium; the presence of both NAC and alpha-tocopherol exerts a synergistic cytoprotection.

2004 - Effetti della N-acetilcisteina in condizioni di stress ossidativo [Abstract in Atti di Convegno]
Iannone, A; Bellei, E; Bergamini, S; Rota, C; Tomasi, A; Albertazzi, A.
abstract

Effetti della N-acetilcisteina in condizioni di stress ossidativo

2004 - Markers of inflammation in end stage renal disease patients. A role for antioxidants [Abstract in Atti di Convegno]
Iannone, A; Bellei, E; Bergamini, S; Rota, C; Tomasi, A
abstract

Markers of inflammation in end stage renal disease patients. A role for antioxidants

2004 - Markers of oxidative stress and antioxidant status in haemodialysis patients [Relazione in Atti di Convegno]
Iannone, A; Bergamini, S; Bellei, E; Rota, C; Lucchi, L; Albertazzi, A; Tomasi, A
abstract

Study of oxidative stress markers and antioxidant status in haemodialysis patients

2004 - Redox regulation and antioxidant in shock [Abstract in Atti di Convegno]
Iannone, A; Bellei, E; Bergamini, S; Rota, C; Tomasi, A
abstract

Redox regulation and antioxidant in shock

2004 - Relationship of asymmetric dimethylarginine to haemodialysis hypotension [Articolo su rivista]
Bergamini, Stefania; L., Vandelli; Bellei, Elisa; Rota, Cristina; P., Manfredini; Tomasi, Aldo; Albertazzi, Alberto; Iannone, Anna
abstract

Hypotension is one of the major complications in patients undergoing haemodialysis (HD), that is well evident in patients defined as hypotension-prone. The mechanisms underlying the hypotensive episodes are not known. We carried out a clinical study on hypotension-prone HD patients to test the existence of a dysregulation in the nitric oxide (NO) generating pathway. Since asymmetric dimethylarginine (ADMA) is an endogenous compound which regulates NO synthesis, we measured its variation in plasma of stable-HD and hypotension-prone patients before, during, and at the end of HD. Before HD, the hypotension-prone patients have higher ADMA levels than stable-HD patients. The HD procedure significantly removes ADMA from plasma of stable-HD patients, while in the hypotension-prone ADMA levels are unchanged at the end of the HD. Moreover, in the hypotension-prone patients, during the hypotensive episode, a dramatic drop of ADMA levels is observed, followed by a rapid increase at the end of the HD. The symmetric dimethylarginine (SDMA), which has no effect on NO synthesis, is also high in plasma of both groups of HD patients compared to normal subjects, and in both groups its levels at the end of HD are significantly reduced. The hypotension-prone patients have basal TNF-alpha levels lower than the stable-HD groups, that significantly increase during the hypotensive episode. On the basis of these findings, we suggest that the hypotensive syndrome could be related to a dysregulation between ADMA metabolism and clearance due both to cytokines release and to an extremely fast ADMA clearance during HD, leading to an increase in NO blood levels.

2004 - Role of nitric oxide generating pathway in haemodialysis hypotension [Poster]
Bergamini, S; Rota, C; Bellei, E; Albertazzi, A; Vandelli, L; Tomasi, A; Iannone, A
abstract

Role of nitric oxide in haemodialysis hypotension

2003 - Measuring oxidative stress and redox state in patients: meaning and perspectives [Poster]
Tomasi, A; Bergamini, S; Bellei, E; Rota, C; Iannone, A.
abstract

Measuring oxidative stress and redox state in patients with ESRD.

2003 - Nitric oxide and hypotension in haemodialysis patients. [Abstract in Rivista]
Iannone, Anna; Bellei, Elisa; Bergamini, Stefania; Rota, C.; Vandelli, L.; Albertazzi, Alberto; Tomasi, Aldo
abstract

proceedings

2003 - Plasma antioxidants modifications during haemodialysis [Poster]
Bellei, E; Manfredini, P; Lucchi, L; Bergamini, S; Rota, C; Tomasi, A; Vandelli, L; Albertazzi, A; Banni, S; Iannone, A.
abstract

The high incidence of atherosclerosis (stroke and ischemic heart disease) in patients with end-stage renal failure (ESRF), associated with clinical and/or laboratory signs of systemic inflammation, has been considered to be in part due to the long-term haemodialysis (HD). The contact with the artificial surface of the extra-corporeal circuit may promote free radicals formation, which is responsible for the increased lipid peroxidation observed in these patients. It has been also demonstrated that they have a decreased intracellular antioxidant capability. To test the presence of oxidative stress and evaluate also the antioxidant status during the dialytic session, we performed a clinical study in a group of functionally anephric patients on maintenance HD treatment with three weekly HD sessions, using a low-flux cellulose diacetate membrane and a freshly prepared bicarbonate buffer.

2003 - “Role of NO in hypotension prone dialytic patients” [Abstract in Rivista]
Iannone, Anna; Bellei, Elisa; Bergamini, Stefania; Rota, Cristina; Vandelli, Lorenza; Albertazzi, Alberto; Tomasi, Aldo
abstract

not available

2002 - Antioxidants modulates benzoyl-peroxide toxicity in human keratinocytes [Abstract in Rivista]
Manfredini, P; Bellei, E; Bergamini, S; Rota, C; Iannone, A; Tomasi, A; Albertazzi, A.
abstract

Benzoyl peroxide (BP) is a free-radical generating compound, widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, BP causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, BP induces DNA strand breaks in many cells, included keratinocytes. In vitro, the tossicity associated with BP is presumably mediated by formation of benzoyloxyl radical, responsable for membrane damage. In this work we investigated the effect of both the lipophilic antioxidant a-tocopherol and the thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to BP.

2002 - “VITAMIN E MODULATES BENZOYL PEROXIDE TOXICITY IN HUMAN KERATINOCYTES” [Relazione in Atti di Convegno]
Manfredini, P; Bellei, Elisa; Bergamini, Stefania; Rota, Cristina; Tomasi, Aldo; Albertazzi, Alberto; Iannone, Anna
abstract

not available

2001 - Cytokine and nitric oxide redox regulation in vivo [Abstract in Atti di Convegno]
Tomasi, A; Bellei, E; Bergamini, S; Rota, C; Iannone, A.
abstract

Inflammation and oxidative stress, at the molecular level, induce peroxidation and modify redox equlibrium.