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ALDO TOMASI

Professore emerito
Facolta' di MEDICINA e CHIRURGIA

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Pubblicazioni

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.

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 - Assessing biocompatibility of face mask materials during covid-19 pandemic by a rapid multi-assays strategy [Articolo su rivista]
Petrachi, T.; Ganzerli, F.; Cuoghi, A.; Ferrari, A.; Resca, E.; Bergamini, V.; Accorsi, L.; Burini, F.; Pasini, D.; Arnaud, G. F.; Piccini, M.; Aldrovandi, L.; Mari, G.; Tomasi, A.; Rovati, L.; Dominici, M.; Veronesi, E.
abstract

During the coronavirus disease 2019 (COVID-19) pandemic, scientific authorities strongly suggested the use of face masks (FMs). FM materials (FMMs) have to satisfy the medical device biocompatibility requirements as indicated in the technical standard EN ISO 10993-1:2018. The biologic evaluation must be confirmed by in vivo tests to verify cytotoxicity, sensitisation, and skin irritation. Some of these tests require an extensive period of time for their execution, which is incompatible with an emergency situation. In this study, we propose to verify the safety of FMMs combining the assessment of 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT) with quantification of nitric oxide (NO) and interleukin-6 (IL-6), as predictive markers of skin sensitisation or irritation based on human primary fibroblasts. Two hundred and forty-two FMMs were collected and classified according to spectrometer IR in polypropylene, paper, cotton, polyester, polyethylene terephthalate, 3-dimensional printing, and viscose. Of all FMMs tested, 50.8% passed all the assays, 48% failed at least one, and only 1.2% failed all. By a low cost, rapid and highly sensitive multi assays strategy tested on human skin fibroblasts against a large variety of FMMs, we propose a strategy to promptly evaluate biocompatibility in wearable materials.

2021 - COVID-19 spreading across world correlates with C677T allele of the methylenetetrahydrofolate reductase (MTHFR) gene prevalence [Articolo su rivista]
Ponti, G.; Pastorino, L.; Manfredini, M.; Ozben, T.; Oliva, G.; Kaleci, S.; Iannella, R.; Tomasi, A.
abstract

Background: Homocysteine assessment has been proposed as a potential predictive biomarker for the severity of COVID-19 infection. The purpose of this review was to analyze the correlation between the prevalence of MTHFR C677 T gene polymorphism and COVID-19 incidence and mortality worldwide. Methods: Data regarding MTHFR C677 T gene mutation were obtained from the interrogation of the Genome Aggregation Database (genomAD), which is publicly available from the web“https://gnomad.broadinstitute.org.” COVID-19 cases, including prevalence and mortality, were obtained from“https://www.worldometers.info/coronavirus” 27 August 2020. Results: There is a clear trend toward the worldwide prevalence of MTHFR 677 T and COVID-19 incidence and mortality. The prevalence of MTHFR 677 T allele in the Latino population, and the incidence and mortality for COVID-19 was higher for this ethnic group than that reported for most other populations globally. Statistical analysis showed a relatively strong correlation between C677 T and death from coronavirus. Conclusions: Genetic polymorphism of MTHFR C677 T may modulate the incidence and severity of COVID-19 pandemic infection.

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 - Homocysteine (Hcy) assessment to predict outcomes of hospitalized Covid-19 patients: A multicenter study on 313 Covid-19 patients [Articolo su rivista]
Ponti, G.; Roli, L.; Oliva, G.; Manfredini, M.; Trenti, T.; Kaleci, S.; Iannella, R.; Balzano, B.; Coppola, A.; Fiorentino, G.; Ozben, T.; Paoli, V. D.; Debbia, D.; De Santis, E.; Pecoraro, V.; Melegari, A.; Sansone, M. R.; Lugara, M.; Tomasi, A.
abstract

2021 - Influence of tooth-brushing on early healing after access flap surgery: A randomized controlled preliminary study [Articolo su rivista]
Bertoldi, C.; Generali, L.; Cortellini, P.; Lalla, M.; Luppi, S.; Tomasi, A.; Zaffe, D.; Salvatori, R.; Bergamini, S.
abstract

In the present study, the clinical outcomes obtained using three different protocols of post-operative plaque control for the 4 weeks after surgery were compared. Thirty healthy subjects, presenting at least one periodontal pocket requiring resective surgery, were selected and randomly distributed to three different groups corresponding to respective post-surgical protocols: (A) toothbrushes + chlorhexidine + anti-discoloration system (ADS + CHX); (B) toothbrushes + chlorhexidine (CHX); (C) only toothbrushes. The full-mouth plaque score (FMPS), full-mouth bleeding score (FMBS), probing pocket depth (PPD), recession depth (REC), clinical attachment level (CAL), and bleeding on probing (BoP) were measured in six aspects per tooth (mesio-buccal (MB), buccal (B), disto-buccal (DB), disto-lingual (DL), lingual (L), and mesio-lingual (ML)) at baseline, 3 months, and 6 months after surgery. FMPS and FMBS did not significantly change (p > 0.05), whereas PPD and CAL significantly decreased, and REC significantly increased in all groups during the study (p < 0.05). Clinical results were satisfactory in all cases, with no significant differences between groups 3 months after surgery. Six months after surgery, only PPD-MB was significantly different in the three groups (p < 0.05). Nevertheless, this value was not clinically relevant because the value of PPD-B (about 2 mm) in group C was physiologic. The mechanical plaque control was proven to be fundamental and sufficient in all the six aspects per tooth to guarantee an excellent clinical outcome without the need of chemical plaque control.

2021 - Liquid biopsy with cell free DNA: new horizons for prostate cancer [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Percesepe, Antonio; Tomasi, Aldo; Ozben, Tomris.
abstract

Although prostate cancer (PCa) is one of the most common tumors in European males, the only minimally invasive diagnostic tool in PCa setup is the determination of PSA in serum. Cell-free DNA (cfDNA) has been demonstrated to be helpful for PCa diagnosis but has not yet been integrated into the clinical setting. This review aims to provide a systematic update of cfDNA and its fragmentation patterns in PCa reported in literature published over the last twenty years. Due to the high variability of the scientific methods adopted and a lack of standardized median cfDNA levels, results fluctuate across different studies. These differences may be due to the cfDNA source, the quantification method, or the fragmentation pattern. Blood plasma is the most frequently analyzed biological fluid, but seminal plasma has been reported to contain higher cfDNA concentration due to its vicinity to the tumor origin. CfDNA has been shown to be composed of single-stranded (ssDNA) and double-stranded DNA (dsDNA), so the total cfDNA concentration should be preferred as it corresponds best to the tumor mass. Fluorometry and capillary electrophoresis (CE) may be quick and cost-effective tools for cfDNA assessment in a clinical setting. The greatest future challenge is the elaboration of common guidelines and standardized procedures for diagnostic laboratories performing cfDNA analysis. A multiparametric approach combining the analysis of total cfDNA (both ssDNA and dsDNA), cfDNA fragment length, and specific genetic mutations (ctDNA assessment) is required for optimal future applications of liquid biopsy.

2021 - Microscopic and chemical characterization of PVC tube used for dialysis lines: A new approach [Articolo su rivista]
Petrachi, T.; Arnaud, G. F.; Roncioni, S.; Resca, E.; Veronesi, E.; Dominici, M.; Tomasi, A.; Cuoghi, A.
abstract

Polyvinylchloride is universally agreed upon to be the material of choice for tubings and for containers for medical application. Many alterations of the chemical/physical surface conditions, mainly due to an altered extrusion process, could influence its biocompatibility by promoting platelet aggregation. Biocompatibility and safety of the medical device must be preserved, also monitoring the migration of additives within polyvinylchloride during the diffusion process. A large variety of methods are used to verify the correct composition and extrusion of polyvinylchloride but, generally, they need long experimental time and are expensive. The aim of the study is to propose a simple, economic and rapid approach based on Fourier transform-infrared spectroscopy and Coomassie Blue staining. The method has been used to detect chemical and morphological defects caused by an altered extrusion process on 20/75 polyvinylchloride tubings in a blind test. This approach positively identified altered samples in 80% of the cases. The suggested approach represents a reliable and versatile method to detect and monitor surface defects by an easy, inexpensive and reproducible method.

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 - Biomarkers associated with COVID-19 disease progression [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Ruini, Cristel; Tomasi, Aldo; Ozben, Tomris
abstract

The SARS-COVID 19 pandemic is a scientific, medical and social challenge. The complexity of the virus is centred on the unpredictable clinical course of the disease that can rapidly develop, causing severe and deadly complications. The identification of effective laboratory biomarkers able to classify patients based on their risk is imperative in being able to guarantee prompt treatment. The analysis of recently published studies highlights the role of systemic vasculitis and cytokine mediated coagulation disorders as the principal actors of multi organ failure in patients with severe COVID-19 complications. The following biomarkers have been identified: haematological (lymphocyte count, neutrophil count, neutrophil-lymphocyte-ratio (NLR), and Hhemoglobin ), inflammatory (C-Reactive Protein (CRP), Erythrocyte Sedimentation Rate (ESR), procalcitonin(PCT )), immunological (IL-6 ) and biochemical (D-Dimer, Troponin, CK, AST ), especially those related to coagulation cascades in disseminated intravascular coagulation (DIC) and acute respiratory distress syndrome (ARDS). New laboratory biomarkers could be identified through the accurate analysis of multicentric case series; in particular, homocysteine and angiotensin II could play a significant role.

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 - Homocysteine as a potential predictor of cardiovascular risk in patients with COVID-19 [Articolo su rivista]
Ponti, G.; Ruini, C.; Tomasi, A.
abstract

2020 - Immunohistochemical mismatch repair proteins expression as a tool to predict the melanoma immunotherapy response [Articolo su rivista]
Ponti, G.; Pellacani, G.; Tomasi, A.; Depenni, R.; Maccaferri, M.; Maiorana, A.; Orsi, G.; Giusti, F.; Cascinu, S.; Manfredini, M.
abstract

In difference to other solid malignancies, the identification of biomarkers for the prediction of malignant melanoma (MM) response to immunotherapy is limited. The aim of the current study was to evaluate the immunohistochemical (IHC) expression of MMR proteins in a cohort of MM metastatic patients receiving anti PD-1 treatments. The therapeutic response of patients was also retrospectively assessed. The cohort of the current study included 14 patients with advanced MM that had received anti PD-1 from January 2014 to December 2016 (12 males, 2 females; average age, 71 years; age range, 47-88 years). IHC analysis of MLH1, PMS2, MSH2 and MSH6 proteins was performed on paraffin-embedded primary tumor samples from each patient and on the 23 available metastasis specimens obtained from the Division of Pathology (University of Modena and Reggio Emilia). The results revealed that 7% of the primary melanoma tissue obtained from the patient cohort exhibited the loss of expression of at least one MMR protein. Three samples from one patient, including one primary melanoma and two metastases, exhibited no MSH6 expression and had the most successful response to anti PD-1 treatment, with a progression-free survival and overall survival of 956 and 2,546 days, respectively. In conclusion, the assessment of MMR protein expression represents a potential predictive marker that may have critical importance for patients with primary and metastatic MM, primarily as criterion for the adoption of immunotherapy treatments.

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.

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 - Non-blood sources of cell-free DNA for cancer molecular profiling in clinical pathology and oncology [Articolo su rivista]
Ponti, G.; Manfredini, M.; Tomasi, A.
abstract

Liquid biopsy can quantify and qualify cell-free (cfDNA) and tumour-derived (ctDNA) DNA fragments in the bloodstream. CfDNA quantification and mutation analysis can be applied to diagnosis, follow-up and therapeutic management as novel oncologic biomarkers. However, some tumor-types release a low amount of DNA into the bloodstream, hampering diagnosis through standard liquid biopsy procedures. Several tumors, as such as brain, kidney, prostate, and thyroid cancer, are in direct contact with other body fluids and may be alternative sources for cfDNA and ctDNA. Non-blood sources of cfDNA/ctDNA useful as novel oncologic biomarkers include cerebrospinal fluids, urine, sputum, saliva, pleural effusion, stool and seminal fluid. Seminal plasma cfDNA, which can be analyzed with cost-effective procedures, may provide powerful information capable to revolutionize prostate cancer (PCa) patient diagnosis and management. In the near future, cfDNA analysis from non-blood biological liquids will become routine clinical practice for cancer patient diagnosis and management.

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.

2019 - Quick assessment of cell-free DNA in seminal fluid and fragment size for early non-invasive prostate cancer diagnosis [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Micali, Salvatore; Torricelli, Federica; Milandri, Riccardo; Del Prete, Chiara; Ciarrocchi, Alessia; Ruini, Cristel; Benassi, Luisa; Bettelli, Stefania; Kaleci, Shaniko; Ozben, Tomris; Tomasi, Aldo
abstract

Liquid biopsy consists in the quantification and qualification of circulating cell-free DNA (cfDNA) and tumor-derived DNA (ctDNA) for cancer recognition. Recently, the characterization of seminal cfDNA (scfDNA) has been reported as a possible biomarker for prostate cancer (PCa) diagnosis.

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 - PTCH1 germline mutations and the basaloid follicular hamartoma values in the tumor spectrum of basal cell carcinoma syndrome (NBCCS) [Articolo su rivista]
Ponti, Giovanni; Manfredini, Marco; Pastorino, Lorenza; Maccaferri, Monia; Tomasi, Aldo; Pellacani, Giovanni
abstract

Background/Aim: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominantly inherited disorder characterized by multiple basal cell carcinomas (BCC), odontogenic tumors and various skeletal anomalies. Basaloid follicular hamartomas (BFHs) constitute rare neoplasms that can be detected in sporadic and familial settings as in the Basaloid Follicular Hamartoma Syndrome (BFHS). Although BFHS shares clinical, histopathological and genetic overlapping with the NBCCS, they are still considered two distinctive entities. The aim of our single-institution study was the analysis of a cohort of PTCH1-mutated patients in order to define clinical and biomolecular relationship between NBCCS and BFHs. Materials and Methods: In our study we evaluated PTCH1 gene-carrier probands affected by NBCCS to detect the incidence of BFHs and their correlation with this rare syndrome. Results: Among probands we recognized 4 patients with BFHs. We found 15 germline PTCH1 mutations, uniformly distributed across the PTCH1 gene. Six of them had familial history of NBCCS, two of them were novel and have not been described previously. Conclusion: NBCCS and BFHS may be the same genetic entity and not two distinctive syndromes. The inclusion of BFH in the NBCCS cutaneous tumor spectrum might be useful for the recognition of misdiagnosed NBCCS cases that could benefit from tailored surveillance strategies.

2018 - Seminal Cell-Free DNA Assessment as a Novel Prostate Cancer Biomarker [Articolo su rivista]
Mandrioli, M.; Manfredini, M.; Micali, S.; Cotugno, M.; Ozben, T.; Pellacani, G.; Tomasi, A.; Ponti, G.; Maccaferri, M.; Bianchi, G.; Del Prete, C.
abstract

Background: Cell-free DNA (cfDNA) includes circulating DNA fragments which can be obtained from different human biological samples. It originates from apoptotic and/or necrotic cells or it is actively secreted by cancer cells. To our knowledge the quantification and size distribution assessment of seminal plasma cfDNA from prostate cancer patients was never assessed. Objective: The aim of our study was to the identify identifation of a novel sensitive non-invasive biomarker of prostate cancer, through the fluorimetric quantification and the electrophoretic analysis of seminal cfDNA in healthy individuals and prostate cancer patients. Results: The concentration of seminal plasma cfDNA in prostate cancer patient was 2243.67 ± 1758 ng/µl. In healthy individuals was 57.7 ± 6.79 ng/µl. Electrophoresis showed broad difference between healthy individuals and patients who showed presented a distinct characteristic DNA ladder fragmentationsmear ranging from 100bp to 2000 10.000 bp. Conclusion: Human seminal fluid can be a valuable source of cfDNA in the setting of liquid biopsy procedures for the identification of novel oncological biomarkers. Seminal plasma cfDNA from prostate cancer patients is significantly more concentrated than from age-matched healthy controls. Fluorimetric measurement and electrophoretic assessment allow a reliable quantification and characterization of seminal plasma cfDNA, which can be used routinely in prostate cancer screening programs.

2018 - Seminal cell free DNA concentration levels discriminate between prostate cancer and benign prostatic hyperplasia [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Micali, Salvatore; Manfredini, Marco; Milandri, Riccardo; Bianchi, Giampaolo; Pellacani, Giovanni; Kaleci, Shaniko; Chester, Johanna; Conti, Andrea; Prete, Chiara Del; Tomasi, Aldo
abstract

Background/Aim: Seminal plasma cfDNA (scfDNA) was recently proposed as a novel PCa biomarker. Our aim was to evaluate whether scfDNA could discriminate PCa from benign prostate hyperplasia (BPH) patients. Patients and Methods: A cohort of 43 patients (18 and 25 pathology proven PCa and BPH patients), and 13 healthy age-matched control subjects were enrolled. scfDNA quantification was performed. Data were analyzed through ANOVA testing. Results: Average scfDNA concentrations were 1,407.83 ng/μl, 128.13 ng/μl and 78.09 ng/μl for PCa patients, BPH patients and healthy subjects, respectively. Statistical analysis showed a significant difference among the groups, allowing for distinction of patients with optimal accuracy. A cut-off level of 450 ng/μl scfDNA was identified for the differentiation of PCa and BPH patients. Conclusion: scfDNA concentrations are significantly different between PCa patients and BPH patients. scfDNA is a promising biomarker with several applications in PCa diagnosis, screening programs and therapeutic monitoring.

2018 - Seminal cell-free DNA assessment as a novel prostate cancer biomarker [Relazione in Atti di Convegno]
Ponti, Giovanni; Maccaferri, Monia; Mandrioli, Mauro; Ozbenc, Tomris; Manfredini, Marco; Micali, Salvatore; Cotugno, Michele; Bianchi, Giampaolo; Pellacani, Giovanni; Tomasi, Aldo
abstract

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2018 - Seminal cell-free DNA molecular profile as a novel diagnostic and prognostic prostate cancer biomarkers [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Cotugno, Michele; Pellacani, Giovanni; Conti, Andrea; Micali, Salvatore; Mandrioli, Mauro; Tomasi, Aldo
abstract

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2018 - The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Kaleci, Shaniko; Mandrioli, Mauro; Pellacani, Giovanni; Ozben, Tomris; Depenni, Roberta; Bianchi, Giampaolo; Pirola, Giacomo Maria; Tomasi, Aldo
abstract

Background Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers. In the present study, the quality and quantity of the cfDNA were assessed by different quantification procedures, in order to identify the potential applications of these techniques in the preliminary cfDNA quantification. Methods Qubit with single (ss) and double strand (ds) DNA assay kits, NanoDrop and quantitative Real Time PCR (qPCR), were adopted to assess the cfDNA in the blood samples of 18 melanoma patients, 67 prostate cancer patients and 15 healthy controls. Results The quantification by NanoDrop (average value 8.48 ng/μl, 95% confidence limit (CL) = 7.23–9.73), Qubit ssDNA (average value 23.08 ng/μl, CL = 19.88–26.28), dsDNA (average value 4.32 ng/μl, CL = 3.52–5.12) assay kits and qPCR (average value 0.39 ng/μl, CL = 0.31–0.47) revealed differences among the four procedures. Qubit 2.0 ss-DNA kit gave higher cfDNA concentration values for all the samples analyzed. In detail, Qubit ssDNA assay revealed higher sensitivity in the quantification of small amounts of pure ss-DNA and ds-DNA, while NanoDrop allowed the assessment of the purity of cfDNA samples. Conclusions The NanoDrop and Qubit 2.0 measurements were analyzed in order to define their correlation with qPCR cfDNA assessment, showing good correlation values with the qPCR that should be considered the “gold standard”. In our proposal, the sequential combination of NanoDrop and Qubit ssDNA methods should be adopted for a cost-effective preliminary assessment of total circulating cfDNA in melanoma and prostate cancer patients, and only discordant values should undergo qPCR assessment.

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 - BRAF ,NRASandC-KITAdvanced Melanoma: Clinico-pathological Features, Targeted-Therapy Strategies and Survival [Articolo su rivista]
Ponti, Giovanni; Manfredini, Marco; Greco, Stefano; Pellacani, Giovanni; Depenni, Roberta; Tomasi, Aldo; Maccaferri, Monia; Cascinu, Stefano
abstract

The mutational status of stage III and IV melanomas should be recognized in order to allow for targeted therapies. The aim of our study was the characterization of BRAF, NRAS and C-KIT melanoma patients, in order to define their optimal management.

2017 - Desmoplastic melanoma: a challenge for the oncologist [Articolo su rivista]
Manfredini, Marco; Pellacani, Giovanni; Losi, Lorena; Maccaferri, Monia; Tomasi, Aldo; Ponti, Giovanni
abstract

To evaluate clinical, pathologic and genetic features of desmoplastic melanoma (DM).MATERIALS & METHODS: Analysis of all DM records from 1991 to 2015. RESULTS: The most common location of DMs was the head and neck (69%); median age and follow-up were 60.5 and 7.3 years, respectively. A familial predisposition for DMs and others malignancies was analyzed. Thin Breslow thickness (<4.5 mm) was associated with an intraepidermal component or a previous lentigo maligna, whereas high Breslow thickness (>4.5 mm) was observed in 'pure' DM. CONCLUSION: DM could progress from an early phase, characterized by an intraepidermal component, to late phase, characterized by a dermal nodule. This hypothesis correlates with melanoma genetic and NF1 mutation, which could be an early event in the progression of DM.

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 - BRAFp.V600E, p.V600K, and p.V600R Mutations in Malignant Melanoma: Do They Also Differ in Immunohistochemical Assessment and Clinical Features? [Articolo su rivista]
Ponti, Giovanni; Tomasi, Aldo; Maiorana, Antonino; Ruini, Cristel; Maccaferri, Monia; Cesinaro, Anna M; Depenni, Roberta; Manni, Paola; Gelsomino, Fabio; Giusti, Francesca; Garagnani, Lorella; Pellacani, Giovanni
abstract

Although the detection of BRAF p.V600E mutation by immunohistochemistry was clearly described in melanoma, discordant evidences were reported for the detection of p.V600K and p.V600R mutations. The aim of the study was to evaluate the efficacy of BRAFp.V600E, p.V600K, and p.V600R detection by immunohistochemistry in melanoma.

2016 - Dynamic thiol/disulphide homeostasis before and after radical prostatectomy in patients with prostate cancer [Articolo su rivista]
Hanikoglu, F.; Hanikoglu, A.; Kucuksayan, E.; Alisik, M.; Gocener, A. A.; Erel, O.; Baykara, M.; Cuoghi, A.; Tomasi, A.; Ozben, T.
abstract

Thiol groups are important anti-oxidants and essential molecules protecting organism against the harmful effects of reactive oxygen species (ROS). The aim of our study is to evaluate thiol–disulphide homeostasis with a novel recent automated method in patients with localized prostate cancer (PC) before and six months after radical prostatectomy (RP). 18 patients with PC and 17 healthy control subjects were enrolled into the study. Blood samples were collected from the controls subjects and patients before and six months after RP. Thiol–disulphide homeostasis was determined using a recently developed novel method. Prostate-specific antigen (PSA), albumin, total protein, total thiol, native thiol, disulphide and total antioxidant status (TAS) were measured and compared between the groups. Native thiol, total thiol and TAS levels were significantly higher in the control group than the patients before RP (p <.001). There was a non-significant increase in the native thiol, total thiol and TAS levels in the patients six months after RP in comparison to the levels before RP (p values.3,.3 and.09, respectively). We found a significant negative correlation between PSA and thiol levels. Our study demonstrated that the decreased thiol and TAS levels weakened anti-oxidant defence mechanism in the patients with PC as indicated. Increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the aetiopathogenesis of prostate cancer.

2016 - Fibroepithelioma of Pinkus: Solitary tumor or sign of a complex gastrointestinal syndrome [Articolo su rivista]
Longo, Caterina; Pellacani, Giovanni; Tomasi, Aldo; Mandel, Victor Desmond; Ponti, Giovanni
abstract

Fibroepithelioma of Pinkus (FEP), which is considered to be an uncommon variant of basal cell carcinoma, has been described in association with other systemic diseases. However, no specific studies are currently available on this subject. The aim of our study was to evaluate the clinical and morphological characteristics of FEP and investigate whether this rare tumor is a single entity or seen in the context of a more complex syndrome. We retrospectively analyzed 49 cases of FEP diagnosed and excised in a single academic institution from 1995 to 2011. The tumors were mainly located on the trunk (77.55%), followed by the lower extremities (12.20%) and the head and neck (10.20%). In 9 of the 49 cases (18%), FEP was associated with gastrointestinal tumors. The abovementioned cases are presented in an attempt to make clinicians more aware of a possible association between FEP and gastrointestinal cancer. Although a possible underlying common genetic background between FEP and gastrointestinal tumors was not provided, our study suggests that patients with FEP should be screened for the occurrence of gastrointestinal tumors.

2016 - Giant elephantiasis neuromatosa in the setting of neurofibromatosis type 1: A case report [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Martorana, Davide; Mandel, Victor Desmond; Loschi, Pietro; Pollio, Annamaria; Pecchi, Annarita; Dealis, Cristina; Seidenari, Stefania; Tomasi, Aldo
abstract

Elephantiasis neuromatosa (EN) can arise from a plexiform neurofibroma of the superficial and deep nerves developing from a hyperproliferation of the perineural connective tissue infiltrating adjacent fat and muscles. To date, the clinical association between EN and neurofibromatosis type 1 (NF1) has been poorly defined, particularly with regard to the role of lymphatic alterations and the consequent lymphedema. The present study reports the clinical and biomolecular features of EN in a NF1 patient with the clear clinical diagnostic criteria of multiple cafè-au-lait macules, neurofibromas, EN, a positive family history and a novel NF1 germline c.1541_1542del mutation. Lymphoscintigraphy (LS) highlighted marked dermal backflow in the affected limb, hypertrophy of the ipsilateral inguinal and external iliac lymph nodes, and a bilateral lower limb lymph flow delay. These data support the hypothesis that an extensive hyperproliferative process involving perineural connective, limb soft tissues, bones and the lymphatic system can be responsible for EN in NF1 patients, on the basis of adipocyte metaplasia triggered by lymphostasis and lymphedema, and bone overgrowth and gigantism caused by chronic hyperemia. LS and magnetic resonance imaging can be efficacious tools in the diagnosis and clinical characterization of the early onset of the disease.

2016 - M09Quantification of circulating cell-free DNA by fluorimetry (Qubit) and spectrophotometry (NanoDrop) in patients with malignant melanoma and prostate cancer [Abstract in Atti di Convegno]
Ponti, G.; Maccaferri, M.; Depenni, R.; Mandrioli, M.; Pellacani, G.; Manfredini, M.; Ozben, T.; Ruini, C.; Iattoni, E.; Cerioli, D.; Cascinu, S.; Tomasi, A.
abstract

Quantification of circulating cell-free DNA by fluorimetry (Qubit) and spectrophotometry (NanoDrop) in patients with malignant melanoma and prostate cancer

2016 - Microsatellite instability, immunohistochemistry and germline mismatch repair gene mutations for the diagnosis of Muir-Torre syndrome in immunosuppressed patients [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Sestini, Roberta; Gorelli, Greta; Tomasi, Aldo
abstract

Although rare sebaceous tumors and keratoacanthomas are clinical criteria for Muir-Torre syndrome (MTS), they can also be found in the context of immunosuppression. We present here two patients who underwent organ transplantation in which immunosuppression unmasked MTS through the early appearance of the cutaneous sebaceous neoplasms. In all of their sebaceous tumors we detected microsatellite instability (MSI) e loss of mutS protein homolog 2 (MSH2) and 6 (MSH6) expression at immunohistochemistry (IHC). Although in the absence of visceral MTS phenotype, we performed the sequencing analysis for mismatch repair genes (MMR) identifying two novel MSH2 and MSH6 germline mutations. The combination of MSI and IHC status can therefore be considered useful for the recognition of MTS, even in case of incomplete MTS phenotype and/or in immunosuppressed patients. It would allow a costeffective approach to identify individuals who should undergo MMR genes direct sequencing.

2016 - Muir-Torre Syndrome and founder Mismatch Repair genes mutations: A long gone historical genetic challenge [Articolo su rivista]
Ponti, Giovanni; Manfredini, Marco; Tomasi, Aldo; Pellacani, Giovanni
abstract

A "cancer predisposing syndrome" later labeled as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) or Lynch syndrome, was firstly described by Warthin, about one century ago. An increased predisposition to the development of multiple familial tumors is described as characteristic of this syndrome where visceral and cutaneous malignancies may appear at an early age namely endometrial, gastric, small bowel, ureteral and renal pelvis, ovarian, hepatobiliary tract, pancreatic, brain (Turcot Syndrome) and sebaceous glands (Muir-Torre Syndrome). The latter, a variant of Lynch Syndrome, is characterized by the presence of sebaceous skin adenomas, carcinomas and/or keratoachantomas associated to visceral malignancies. Both Lynch Syndrome and Muir-Torre Syndrome have been recognized as due to germline mutations in mismatch repair genes MLH1, MSH2 and MSH6. To date, 56 Lynch Syndrome founder mutations dependent on MLH1, MSH2 and, although less frequently found, MSH6 and PMS2 are described. Some of these founder mutations, principally of MSH2 gene, have been described to cause Muir-Torre phenotype and have been traced in large and outbreed Muir-Torre Syndrome families living in different US and European territories. Due to the evidences of highly specific Muir-Torre phenotypes related to the presence of widespread MSH2 founder mutations, preliminary search for these MSH2 common mutations in individuals carring sebaceous tumors and/or keratoacanthomas, at early age or in association to visceral and familial tumors, permits cost-effective and time-saving diagnostics strategies for Lynch/Muir-Torre syndromes.

2016 - Oral mucosal stigmata in hereditary-cancer syndromes: From germline mutations to distinctive clinical phenotypes and tailored therapies [Articolo su rivista]
Ponti, Giovanni; Tomasi, Aldo; Manfredini, Marco; Pellacani, Giovanni
abstract

Numerous familial tumor syndromes are associated with distinctive oral mucosal findings, which may make possible an early diagnosis as an efficacious marker for the risk of developing visceral malignancies. In detail, Familial Adenomatous Polyposis (FAP), Gardner syndrome, Peutz-Jeghers syndrome, Cowden Syndrome, Gorlin Syndrome, Lynch/Muir-Torre Syndrome and Multiple Endocrine Neoplasia show specific lesions of the oral mucosa and other distinct clinical and molecular features. The common genetic background of the above mentioned syndromes involve germline mutations in tumor suppressor genes, such as APC, PTEN, PTCH1, STK11, RET, clearly implied in both ectodermal and mesodermal differentiation, being the oral mucosal and dental stigmata frequently associated in the specific clinical phenotypes. The oral and maxillofacial manifestations of these syndromes may become visible several years before the intestinal lesions, constituting a clinical marker that is predictive for the development of intestinal polyps and/or other visceral malignancies. A multidisciplinary approach is therefore necessary for both clinical diagnosis and management of the gene-carriers probands and their family members who have to be referred for genetic testing or have to be investigated for the presence of visceral cancers.

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.

2016 - Quantification of circulating cell-free DNA by fluorimetry (Qubit) and spectrophotometry (NanoDrop) in patients with malignant melanoma and prostate cancer [Abstract in Rivista]
Ponti, Giovanni; Maccaferri, M; Depenni, Roberta; Mandrioli, Mauro; Pellacani, Giovanni; Manfredini, M; Ozben, T; Ruini, C; Iattoni, Elena; Cerioli, Davide; Cascinu, Stefano; Tomasi, Aldo
abstract

n.d.

2016 - Role of microsatellite instability, immunohistochemistry and Mismatch Repair germline aberrations in immunosuppressed transplant patients: a phenocopy dilemma in Muir-Torre Syndrome. [Articolo su rivista]
Ponti, Giovanni; Manfredini, Marco; Pellacani, Giovanni; Tomasi, Aldo
abstract

Sebaceous tumours and keratoacanthomas are uncommon neoplasms that constitute important clinical criteria for Muir-Torre Syndrome (MTS) diagnosis. In MTS patients, the increased risk of developing synchronous or metachronous visceral malignancies is characterized by autosomal dominant inheritance. However, there are further conditions, other than MTS, that increase the risk of sebaceous neoplasms, e.g. iatrogenic immunosuppression. In this latter scenario, the sebaceous tumours can present Microsatellite instability (MSI) and loss of Mismatch-Repair (MMR) proteins, characteristic of hereditary syndromes, even in the absence of MMR germline mutations. In this paper we examine transplant probands in which the immunosuppressive therapies unmask the MTS cutaneous phenotypes, showing microsatellite instability (MSI) and loss of MMR protein expression, as demonstrated by immunohistochemistry (IHC). Furthermore, mismatch repair genes (MMR) sequencing analysis identified the presence of germline mutations in MTS-suspected individuals, in the absence of a visceral MTS phenotype. It is well known that immunosuppression plays a central role in the development of sebaceous tumours in both MTS and in non-syndromic settings. Sebaceous skin tumours MSI status and IHC profiles can be influenced by epigenetic or iatrogenic factors, however they constitute valuable tools and a cost-effective approach to screen individuals who otherways should undergo MMR genes direct sequencing in the context of immunosuppression. In this complex setting, the choice of the immunosuppressive drug becomes a critical decision for the management of both MTS and sporadic transplant patients, that may benefit from the administration of immunosuppressive drugs, resulting in a low impact on skin cancerogenesis.

2016 - Skeletal stigmata as keys to access to the composite and ancient Gorlin-Goltz syndrome history: The Egypt, Pompeii and Herculaneum lessons [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Tomasi, Aldo; Sammaria, Giuliano; Manfredini, Marco
abstract

There are several genetic diseases with a wide spectrum of congenital bone stigmata in association to cutaneous and visceral benign and malignant neoplasms. Gorlin-Goltz syndrome, also named nevoid basal cell carcinoma syndrome, is an autosomal dominant systemic disease with almost complete penetrance and high intra-familial phenotypic variability, caused by germline mutations of the gene PTCH1. The syndrome is characterized by unusual skeletal changes and high predisposition to the development of multiple basal cell carcinomas, odontogenic keratocysts tumors and other visceral tumors. The Gorlin syndrome, clinically defined as distinct syndrome in 1963, existed during Dynastic Egyptian times, as revealed by a costellation of skeletal findings compatible with the syndrome in mummies dating back to 3000years ago and, most likely, in the ancient population of Pompeii. These paleogenetic and historical evidences, together with the clinical and biomolecular modern evidences, confirm the quite benign behavior of the syndrome and the critical value of the multiple and synchronous skeletal anomalies in the recognition of these rare and complex genetic 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 - Confocal microscopy characterization of BRAFV600E mutated melanomas [Articolo su rivista]
Ruini, Cristel; Manfredini, Marco; Pellacani, Giovanni; Mandel, Victor Desmond; Tomasi, Aldo; Ponti, Giovanni
abstract

Thanks to modern techniques, molecular signatures for melanoma are now identifiable and have opened new horizons in the treatment of metastatic disease with molecular-targeted therapies. We distinguish different melanoma subtypes on the basis of genetic mutations such as BRAFV600E and we can therefore hypothesize the existence of corresponding morphological patterns that might be detected in vivo by noninvasive diagnostic tools such as dermoscopy and confocal microscopy. Eight BRAFV600E mutated melanomas (six primary and two metastases) were collected, matched in terms of age, sex, and thickness wild-type controls, and analyzed. In this preliminary study, regression, corresponding to fibrosis and melanophages in the dermis, was the predominant pattern and was also observed confocally when dermoscopy showed no peppering. In particular, confocal microscopy could not only detect regression but also provided a semiquantitative analysis of its grade through the count of melanophages. Confocal microscopy can be proposed as a useful tool in the preliminary screening and characterization of BRAFV600E mutated melanomas, providing new insights for patients' screening and follow-up.

2015 - Distinctive clinical and dermoscopic features of BRAFp.V600K mutated melanomas. [Articolo su rivista]
Ponti, Giovanni; Manfredini, M; Tomasi, Aldo; Pellacani, Giovanni
abstract

dermoscopic criteria associated with BRAF and NRAS mutation status in primary cutaneous melanoma have been recently analyzed by Pozzobon et al. in an interesting study published in your journal. The authors found out that peppering pattern was significantly increased among BRAF mutant melanoma, being present in the 68% of their cases series, with OR 1.68 (CI 95%1.089-2.581, p=0.015).1 The intend of this letter is to underline the dermoscopic features and the distinctive clinical features of BRAF p.V600K melanoma (the second most common BRAF V600 mutation detected in 5 to 30% of cases), that might be slightly different from from BRAF p.V600E melanoma.

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 - Fordyce granules and hyperplastic mucosal sebaceous glands as distinctive stigmata in Muir-Torre syndrome patients: characterization with reflectance confocal microscopy [Articolo su rivista]
Ponti, Giovanni; Meschieri, Andrea; Pollio, Annamaria; Ruini, Cristel; Manfredini, Marco; Longo, Caterina; Mandel, Victor Desmond; Ciardo, Silvana; Tomasi, Aldo; Giannetti, Luca; Pellacani, Giovanni
abstract

Background: The Muir-Torre syndrome (MTS), a variant of Lynch syndrome (LS), is characterized by the presence of sebaceous skin adenomas and/or carcinomas and keratoacanthomas associated with visceral malignancies. Fordyce granules (FGs) are oral mucosal lesions previously found in association with LS. The aim of this study was to analyze the specific frequency of FGs in sporadic individuals and gene carriers patients with MTS of known mismatch repair genes mutations. The secondary aim was to characterize FGs by means of reflectance confocal microscopy (RCM). Methods: A total of 13 patients belonging to nine different genetically unrelated MTS kindreds (MLH1 gene mutation n = 2; MSH2 gene mutation n = 11) and 140 genetically unrelated healthy controls were examined. Depending on the clinical examination of the oral mucosa surface, subjects were categorized as either FGs positive or FGs negative. Results: FGs were diagnosed in 13 of 13 (100%) of MMR gene carriers patients with MTS vs. 9 of 140 (6.4%) controls. The most common site for FGs in MTS was the vestibular oral mucosa, compared with the gingival mandibular and retromandibular pad in controls. RCM examination found multiple sebaceous acinar cells that appear as round or oval hyper-refractive globules and that create a lobular aspects of the sebaceous glands defined as 'moruliform' or 'berry-like' structures. Conclusions: Clinical and RCM evidences of our study suggest that an activation of the sebaceous glands system occurs in patients with MTS. Fordyce granules and intra-oral sebaceous hyperplasia may constitute an additional clinical parameter, which may be adopted to distinguish individuals with highest likelihood of being affected from MTS.

2015 - Giuseppe Moscati: a man, a physician and a scientist [Articolo su rivista]
Ponti, Giovanni; D'Onofrio, Felice; Ruini, Cristel; Muscatello, Umberto; Tomasi, Aldo
abstract

The life of Giuseppe Moscati (1880-1927) as a man, as a physician and as a scientist may be framed within the cultural climate of Positivism, which spread over the last years of the 19th century and the beginning of the 20th Century. His activity contributed to patients' care improvement; in addition to meticulous drug regimens, he also prescribed a methodology of spiritual care, involving meditation and self-control as part of an holistic approach to healthcare. Our review deals with his published researches, highlighting the innovative findings on the juvenile diabetes treatment and extensive clinical changes consequent upon nephritis. This extraordinary man put considerable emphasis on primary care and holistic health in Italy, pioneering a new patient-centred, and holistic approach to medicine.

2015 - Mismatch Repair Gene Deficiency and Genetic Anticipation in Lynch Syndrome [Articolo su rivista]
Ponti, Giovanni; Ruini, Cristel; Tomasi, Aldo
abstract

Lynch syndrome; mismatch repair genes

2015 - Mismatch repair genes founder mutations and cancer susceptibility in Lynch syndrome [Articolo su rivista]
Ponti, Giovanni; E., Castellsagué; C., Ruini; Percesepe, Antonio; Tomasi, Aldo
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 - 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 - 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 - Brooke-Spiegler syndrome tumor spectrum beyond the skin: a patient carrying germline R936X CYLD mutation and a somatic CYLD mutation in Brenner tumor [Articolo su rivista]
Ponti, Giovanni; Ruini, Cristel; Girolomoni, Giampiero; Pellacani, Giovanni; Farnetani, Francesca; Pastorino, Lorenza; Ghiorzo, Paola; Witkowski Alex, M.; Bianchi Scarrà, Giovanna; Tomasi, Aldo; Loschi, Pietro; Nasti, Sabina
abstract

Brooke–Spiegler syndrome is a hereditary disorder characterized by predisposition to the development of skin appendage tumors and the major and minor salivary glands neoplasms. The role of the CYLD mutation in visceral neoplasms is still unclear, except for parathyroid tumor. We report the case of a 46 year-old patient with multiple cylindromas and trichoepiteliomas, a Brenner tumor of the ovary, and a negative family history for Brooke-Spiegler phenotype. Genetic analysis revealed R936X germline mutation in the proband but not in her relatives. The same somatic mutation was found in the Brenner tumor, together with a novel missense CYLD mutation (D889N), which has never been reported in the literature to our knowledge. A founder effect for R936X has been hypothesized due to its high; surprisingly in our case this mutation seems to be recognized as a de novo mutation. Future studies involving a greater number of cases are necessary to understand possible genotype/phenotype correlations, through the clinical analysis of the familial tumor spectrum.

2014 - Erratum: Molecular targeted approaches for advanced BRAF V600, N-RAS, c-KIT, and GNAQ melanoma (Disease Markers) [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Tomasi, Aldo; Loschi, Pietro; Luppi, Gabriele; Gelsomino, Fabio; Longo, Caterina
abstract

ND

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 - Giuseppe Moscati (1880-1927): a holistic approach to medicine [Articolo su rivista]
Ponti, Giovanni; Tomasi, Aldo
abstract

Giuseppe Moscati was a physician, medical school professor and a pioneer in the field of biochemistry and Italian studies on diabetes. He was declared a Catholic saint in 1987. In order to respond better to both the physical and spiritual needs of his patients, he developed his own holistic approach to healthcare involving meticulous drug regimens, meditation and discipline.

2014 - Hypomelanosis of Ito with a trisomy 2 mosaicism: a case report [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Tomasi, Aldo; Percesepe, Antonio; Guarneri, Carmelo; Guerra, Azzurra; Mandel, Victor Desmond; Kisla, Elif; Cevikel, Piril; Neri, Claudia; Menozzi, Cristina; Seidenari, Stefania
abstract

Introduction: Hypomelanosis of Ito is a rare neurocutaneous disorder, characterized by streaks and swirls of hypopigmentation following the lines of Blaschko that may be associated to systemic abnormalities involving the central nervous system and musculoskeletal system. Despite the preponderance of reported sporadic hypomelanosis of Ito, few reports of familial hypomelanosis of Ito have been described. Case presentation: A 6-month-old Caucasian girl presented with unilateral areas of hypomelanosis distributed on the left half of her body and her father presented with similar mosaic hypopigmented lesions on his upper chest. Whereas both blood karyotypes obtained from peripheral lymphocyte cultures were normal, a 16% trisomy 2 mosaicism was found in cultured skinfibroblasts derived from a hypopigmented skin area of her father. Conclusions: Familial cases of hypomelanosis of Ito are very rare and can occur in patients without systemic involvement. Hypomelanosis of Ito constitutes a non-specific diagnostic definition including different clinical entities with a wide phenotypic variability, either sporadic or familial. Unfortunately, a large number of cases remain misdiagnosed due to both diagnostic challenges and controversial issues on cutaneous biopsies in the pediatric population.

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 - Molecular Targeted Approaches for Advanced BRAF V600, N-RAS, c-KIT, and GNAQ Melanomas [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Tomasi, Aldo; Loschi, Pietro; Luppi, Gabriele; Gelsomino, Fabio; Longo, Caterina
abstract

The introduction of a newly developed target therapy for metastatic melanomas poses the challenge to have a good molecular stratification of those patients who may benefit from this therapeutic option. Practically, BRAF mutation status (V600E) is commonly screened although other non-V600E mutations (i.e., K-R-M-D) could be found in some patients who respond to therapy equally to the patients harboring V600E mutations. Furthermore, other mutations, namely, N-RAS, KIT, and GNAQ, should be sequenced according to distinct melanoma specific subtypes and clinical aspects. In our report, a practical flow chart is described along with our experience in this field.

2014 - Muir-Torre syndrome or phenocopy? The value of the immunohistochemical expression of mismatch repair proteins in sebaceous tumors of immunocompromised patients [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Ruini, Cristel; Percesepe, Antonio; Longo, Caterina; Mandel, Victor Desmond; Crucianelli, Francesca; Gorelli, Greta; Tomasi, Aldo
abstract

Primary and secondary immunodepressive conditions are associated with an increased incidence of sebaceous tumors. Microsatellite instability (MSI) and lack of expression of mismatch repair (MMR) proteins, typical markers of Muir-Torre/Lynch heredo-familial settings, can be recognized also in immunocompromised patients. We aimed to carry on a systematic examination of clinical, immunohistochemical, biomolecular features of sebaceous tumors arising in immunocompromised and immunocompetent patients between 1986 and 2012. Microsatellite screening, immunohistochemical analysis and genetic testing were performed for hMLH1, hMSH2 and hMSH6. Methylation status of MMR genes was checked in cases with immunohistochemistry (IHC) loss of MMR proteins expression and no germline mutations. Fifteen patients had a personal history of visceral carcinomas fulfilling diagnostic criteria for Muir-Torre syndrome. In this cohort, IHC analysis, MSI status and genetic testing were in agreement, showing eight MSH2 and two MLH1 germline mutations. Five patients were immunosuppressed and their sebaceous tumors showed a lack of MSH2/MSH6 expression, although just one case with positive family history for visceral cancer harbored a germline mutation. In immunosuppressed patients, loss of IHC for MMR proteins is not necessarily secondary to MMR germline mutations. IHC false positives are probably due to epigenetic alterations. MSI and lack of expression of MMR proteins can be recognized also in immunocompromised patients without MMR germline mutations.

2014 - Multiple primary melanomas versus single melanoma of the head and neck: a comparison of genetic, diagnostic, and therapeutic implications [Articolo su rivista]
Pollio, Annamaria; Tomasi, Aldo; Pellacani, Giovanni; Ruini, Cristel; Mandel, Victor Desmond; Fortuna, Giulio; Seidenari, Stefania; Ponti, Giovanni
abstract

Single primary and multiple primary melanomas (MPMs) of the head and neck region may be confused at first glance because of the common clinical and dermoscopic patterns. An inaccurate diagnosis may lead the clinician to a wrong diagnostic and therapeutic pathway because MPMs occurring in familial or sporadic settings are often involved in individual cancer susceptibility. We investigated the clinical, demographic, histological, and survival differences between MPMs and single melanoma occurring in the head and neck region. A retrospective analysis of medical and histologic records from 217 melanomas of the head and neck region was carried out. Malignant neoplasms affecting MPMs patients were also reported. Mutational analysis of specific genes was carried out when clinical data and family history were suggestive for a familial/hereditary setting. Two hundred and five out of 217 (94.5%) patients were affected by single primary melanoma and 12 (5.5%) by MPMs of the head and neck region. Individuals affected by MPMs were distinguished by a significantly higher mutation frequency and a higher prevalence of malignant neoplasms such as renal cancer. Genetic testing showed germline mutations affecting MITF E318K, CDKN2A genes. Our data highlight the importance of strict cancer surveillance in individuals with MPMs and the role of appropriate genetic counseling and testing in selected patients. Finally, personalized clinical and instrumental screening and follow-up strategies should also be based on mutational status. A heightened level of suspicion is required in the clinical management of mutation carriers.

2014 - NF1 truncating mutations associated to aggressive clinical phenotype with elephantiasis neuromatosa and solid malignancies [Articolo su rivista]
Ponti, Giovanni; Martorana, Davide; Pellacani, Giovanni; Ruini, Cristel; Loschi, Pietro; Baccarani, Alessio; DE SANTIS, Giorgio; Pollio, Annamaria; Tauro, Maria Neri; Mandel, Victor Desmond; Maiorana, Antonio; Maccio, Livia; Maccaferri, Monia; Tomasi, Aldo
abstract

Background/aim: Von Recklinghausen disease is a syndrome characterized by a wide phenotypic variability giving rise to both, cutaneous and visceral benign and malignant neoplasms. The first include cutaneous neurofibromas, subcutaneous and plexiform neurofibromas. The latter can undergo malignant transformation and/or determine elephantiasis neuromatosa. Visceral tumors may include malignant peripheral nerve sheet tumors, gastrointestinal stromal tumors, cerebral gliomas and abdominal neurofibromas. In the present study, the authors discuss the clinical and biomolecular characterization of a cohort of 20 families with a diagnosis of type 1 neurofibromatosis. Patients and methods: Clinically, the cohort includes three probands with elephantiasis neuromatosa and a peculiarly high incidence of breast and gastrointestinal cancer. Results: Among the 14 NF1 mutations documented, 10 encoding for a truncated protein have been associated to particularly aggressive clinical phenotypes including elephantiasis neuromatosa, malignant peripheral nerve sheet tumors, breast cancer, gastrointestinal stromal tumors. Conclusion: This effect on protein synthesis, rather than the type of NF1 mutation, is the key to the explanation of the genotype-phenotype correlations in the context of neurofibromatosis type 1.

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 (&) ? 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 - Skeletal and cranio-facial signs in Gorlin syndrome from ancient Egypt to the modern age: Sphenoid asymmetry in a patient with a novel PTCH1 mutation [Articolo su rivista]
Ponti, Giovanni; Ruini, Cristel; Pastorino, Lorenza; Loschi, Pietro; Pecchi, Annarita; Malagoli, Marcella; Mandel, Victor Desmond; Boano, Rosa; Conti, Andrea; Pellacani, Giovanni; Tomasi, Aldo
abstract

Gorlin syndrome is an autosomal dominant disorder linked to PTCH1 mutation, identified by a collection of clinical and radiologic signs. We describe the case of a family in which father and son fulfilled clear cut diagnostic criteria for Gorlin syndrome including multiple basal cell carcinomas, keratocystic odontogenic tumors, atypical skeletal anomalies and a novel PTCH1 germline mutation (c.1041delAA). Craniofacial and other skeletal anomalies displayed at 3D and helical CT scan were: macrocephaly, positional plagiocephaly, skull base and sphenoid asymmetry, bifidity of multiple ribs and giant multilocular odontogenic jaw cysts. Extensive multilamellar calcifications were found in falx cerebri, tentorium, falx cerebelli and in the atlanto-occipital ligament. The inclusion of bifid ribs as a novel major criteri may be useful for the recognition and characterization of misdiagnosed cases.

2014 - Stem cell properties in cell cultures from different stage of melanoma progression. [Articolo su rivista]
Magnoni, Cristina; S., Guidice; Pellacani, Giovanni; G., Bertazzoni; Longo, Caterina; E., Veratti; D., Morini; L., Benassi; S., Al Jalbout; C., Vaschieri; P., Azzoni; DE POL, Anto; S., Seidenari; Tomasi, Aldo; Ponti, Giovanni
abstract

Cutaneous Melanoma is an extremely heterogeneous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesions and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchimal transition (EMT). The aim of this study was to clarify the role of stem cell-like population in human melanomas through the in vitro analysis of melanocytic cell cultures, obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma larger than 2cm diameter and/or wider than 300mm2 surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo melanoma (LM), superficial spreading (SS), nodular melanoma (NM) and metastatic melanoma (MM) and maintained in different media in order to evaluate the colony-forming unit assay, alkaline phosphatase and toluidine blue stain and the cell ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed in order to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166 and nestin. This study confirms that melanoma can include an heterogeneous cell population with both abilities to self-renew and to give rise to differentiated progeny. Melanoma cells displayed the intra-tumoral heterogeneity and dynamic antigen phenotypes. Histologically, the transitions from normal skin to DN to LM to NM to MM was associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin and CD73 by melanoma cells. These molecular evidences could be a milestone for the development of novel biomolecular targeted-therapy approaches.

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 - Are the neck malignant melanomas different from the ones affecting the head? Clinicopathologic, dermoscopic and prognostic findings [Articolo su rivista]
Ponti, Giovanni; Pollio, Annamaria; Tomasi, Aldo; Borsari, Stefania; Magnoni, Cristina; Mandel, Victor Desmond; Loschi, Pietro; Ruini, Cristel; Seidenari, Stefania
abstract

Background: Malignant melanomas of the head and neck are usually considered as a unique entity in comparison to other body sites. However, no characterization of neck melanoma has been performed so far, despite the clear anatomic and histological differences. Aim: We investigated clinical, demographic, histological and dermoscopic differences between face, scalp and neck melanoma. Materials and methods: A retrospective analysis of medical and histologic records from 116 melanomas of the head and neck area collected between January 2003 and January 2008 was performed. Body site, gender, age, number of lesions, age at first melanoma diagnosis, size, Clark level, association with nevi, presence or absence of mitoses and/or ulceration, presence of synchronous and/or metachronous melanoma were recorded. Moreover, digital dermoscopy images of 92 melanomas of the head and neck area were analyzed for main dermoscopic patterns and lesion diameter. Results: Significant differences in Breslow thickness, ex-naevo origin and tumor size among neck and face-scalp melanomas were observed. Neck MM patients were younger than those with MM of face and scalp. In contrast to scalp and face, no patient died from neck melanoma. Dermoscopic patterns were similar to those of trunk-limbs MM, and no lesion showed a lentigo maligna pattern which was observed in most lesions of the face. Conclusion: Melanomas of the neck must be distinguished from face and scalp melanomas because of younger age, different dermoscopic patterns and ex-naevo origin and better prognosis. These data should be taken into account both from an epidemiological and clinical point of view.

2013 - Cancer-associated genodermatoses: Skin neoplasms as clues to hereditary tumor syndromes. [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; S., Seidenari; A., Pollio; Muscatello, Umberto; Tomasi, Aldo
abstract

Characteristic skin neoplasms are associated with a large number of hereditary tumor syndromes and their knowledge and early detection may facilitate the diagnosis of the underlying malignancies. We will review the clinical and dermatopathological aspects of cutaneous and visceral lesions and the recent progresses in understanding the etiology, pathogenesis and therapies of selected tumor syndromes. The skin neoplasms we chose to consider are multiple neurofibromas in neurofibromatosis, cylindromas and trichoepitheliomas in Broke-Spiegler syndrome, sebaceous tumors and keratoacanthomas in Muir-Torre syndrome, Gardner fibromas in Gardner syndrome, multiple basal cell carcinomas in nevoid basal cell carcinoma (Gorlin) syndrome, multiple tricholemmomas in Cowden syndrome, multiple fibrofolliculomas in Birt-Hogg-Dubé syndrome and multiple leiomyomas in hereditary leiomyomatosis and renal cell cancer. Hereditary cancers have distinct biological and clinical features as compared to their sporadic counterparts; for this reason, we are now able to experiment new treatment approaches involving not only tumor detection and prevention, but also tailored therapeutic strategies focusing on the peculiar druggable molecular targets.

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 - Fluorescence in-situ hybridization and dermoscopy in the assessment of controversial melanocytic tumors. [Articolo su rivista]
Ponti, Giovanni; Ruini, C; Massi, D; Pellacani, Giovanni; Tomasi, Aldo; Paglierani, M; Loschi, Pietro; Seidenari, Stefania
abstract

Although the 'gold standard' for melanoma diagnosis remains histopathological analysis, presently dermoscopists play a significant role in the diagnostic process. However, even a combined approach may not allow a clear-cut judgment on equivocal melanocytic lesions. Fluorescence in-situ hybridization (FISH) can offer assistance in the evaluation of chromosome abnormalities associated with malignancies, and its role is emerging in melanoma diagnosis. The aim of this study was to evaluate the diagnostic role of the FISH in the assessment of controversial lesions, defined as those lesions showing discrepancies between dermatoscopic and histological evaluations. Twenty clinically and histologically ambiguous melanocytic lesions were selected. After the first histopathologic diagnosis, a second pathologist examined the specimens in a blinded review for a second opinion and to identify the most suitable areas to hybridize using probes specific to RREB1, MYB, and CCND1 genes and the centromere of chromosome 6. The first histopathological evaluation led to the diagnosis of melanoma in seven cases, whereas the second identified eight cases of malignant melanoma and was in agreement with the first in 65% of cases and with dermoscopy in 40% of cases. Cytogenetic abnormalities detected by FISH are markers of malignancy that can be useful in the characterization of difficult-to-diagnose melanocytic tumors, when the dermatologist and the pathologist have a different opinions.

2013 - Hereditary trichilemmal cysts: a proposal for the assessment of diagnostic clinical criteria [Articolo su rivista]
Seidenari, Stefania; Pellacani, Giovanni; Nasti, Sabina; Tomasi, Aldo; Pastorino, Lorenza; Ghiorzo, Paola; Ruini, Cristel; BIANCHI-SCARRÀ, Giovanna; Pollio, Annamaria; Mandel, Victor Desmond; Ponti, Giovanni
abstract

Trichilemmal cysts (TCs) can occur as sporadic lesions or in hereditary-familial settings with autosomal dominant transmission. These entities have not been widely analyzed in their peculiar aspects yet. The aim of this study was to describe a cohort of patients with diagnosis of TCs through a clinical and biomolecular characterization, intended to highlight some effective diagnostic criteria for their identification. Among 149 cases of this study, 24 cases of TCs (16.1%) arose in patients with at least one first-degree relative with diagnosis of TCs. Peculiar findings concerning hereditary lesions included the multiple presentation with an early onset age. On the basis of clinical evaluation, we propose a panel of clinical and histologic criteria for the diagnosis of hereditary TCs, which includes: (i) the diagnosis of TCs in at least two first-degree relatives or in three first- or second-degree relatives in two consecutive generations; (ii) at least one of the patients with TCs diagnosed <45 years; and (iii) the diagnosis of multiple or giant (>5-cm lesions) or rare histopathologic features (proliferating and ossifying) TCs.

2013 - Malignant and benign tumors associated to multiple primary melanomas: just the starting block for the involvement of MITF, PTEN and CDKN2A in multiple cancerogenesis? [Articolo su rivista]
A., Pollio; Tomasi, Aldo; Seidenari, Stefania; Pellacani, Giovanni; M., Rodolfo; S., Frigerio; A., Maurichi; D., Turchetti; S., Bassoli; C., Ruini; Ponti, Giovanni
abstract

Background: Multiple primary melanomas (MPMs) could represent an interesting field of investigation for possible linkage between multiple cancer phenotypes and genetic background. In this setting, the co-existence of MPMs and malignant/benign neoplasms in a group of MPMs patients has been highlighted and the corresponding germ-line mutations have been traced. Methods: The study evaluated the prevalence of benign and malignant neoplasms in a group of 49 MPMs patients and compared the data with 49 age- and gender-matched single primary melanoma (SPM) controls. Genetic testing was performed when a germ-line mutation was suspected. Results: A statistically significant prevalence (P<0.0001) of benign and malignant neoplasms were found among MPMs patients. Of 27 diagnosed malignancies, basal cell carcinoma was the most frequent (n=10, 37.1%), followed by colorectal adenocarcinoma (n=4, 14.8%), prostate adenocarcinoma (n=3, 11.1%), breast adenocarcinoma (n=2, 7.4%), papillary thyroid carcinoma (n=2, 7.4%), pancreas adenocarcinoma (n=2, 7.4%), renal cell carcinoma (n=2, 7,4%), oral squamous carcinoma (n=1, 3.7%), liver adenocarcinoma (n=1, 3.7%), large B lymphoma (n=1, 3.7%), multiple myeloma (n=1, 3.7%), urinary bladder carcinoma (n=1, 3.7%), stomach carcinoma (n=1, 3.7%). Ten different benign neoplasms arising in various organs were also reported. Germline mutations involving PTEN, MITF E318K, CDKN2A, MC1R were detected. Conclusions: Close cancer surveillance should be recommended in MPMs patients. Clinicians should select the appropriate population for genetic testing and refer those patients for genetic counseling. Tailored clinical and instrumental screenings and follow-up strategies have to be based on the patient’s mutation status.

2013 - Multiple tumor phenotypes and malignant melanoma: the role of genetic testing for MITF, PTEN and CDKN2A. [Abstract in Atti di Convegno]
Ponti, Giovanni; Depenni, R; Luppi, G; Ruini, C; Gelsomino, F; Loschi, P; Tomasi, Aldo; Spallanzani, A; Pellacani, Giovanni
abstract

It is well known that synchronous and/or metachronous multiple primary melanomas (MPMs) occur in clearly hereditary settings as well as in familial and sporadic settings. However, no evidence exists about their co-occurrence in patients with multiple cancer phenotypes, a setting in which genetic susceptibility plays a significant role. Inside the multiple tumor phenotypes, multiple primary melanomas occurring in familial or sporadic settings constitute an interesting case study for the analysis of cancer susceptibility. In this study, we focused on the co-existence of MPMs and other types of tumors evaluating the genetic characterization underlying the context of high susceptibility to the development of multiple cancer phenotypes. We retrospectively evaluated the prevalence of benign and malignant neoplasms occurred in a group of 49 patients with multiple primary melanomas (MPMs) and compared the results with a group of 58 randomly age- and gender matched controls with single primary melanoma (SPM) and a group of 52 age- and gender randomly matched healthy patients. Mutational analysis of specific genes was performed when clinical data and family history were suggestive for familial/hereditary setting. Individuals affected by MPMs were distinguished by a statistically significant higher mutation frequency and a higher prevalence of other neoplasms. Of 27 diagnosed malignancies, basal cell carcinoma was the most frequent (n=10, 37.1%), followed by colorectal adenocarcinoma (n=5, 18.5%) and prostate adenocarcinoma (n=3, 11.1%). In addition to malignances, we also detected 10 benign tumors. Genetic testing revealed germline mutations affecting PTEN, MITF E318K, CDKN2A, MC1R genes. Our data highlight the importance of strict cancer surveillance in individuals with MPMs and the role of appropriate genetic counseling and testing in selected patients. Selected candidates should undergo genetic testing, not only for CDKN2a, CDK4, MC1R, but also for MITF E318K and PTEN, in ordeto plan personalized clinical and instrumental screenings and follow-up strategies; the latter must be assessed basing on mutational status: it is prudent to have a heightened level of suspicion in the clinical management of mutation carriers.

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 - Relationship between histological and computer based assessment of melanoma diameter and thickness in head & neck vs. trunk melanoma. [Articolo su rivista]
Seidenari, Stefania; A., Fabiano; S., Al Jalbout; S., Bassoli; S., Borsari; Magnoni, Cristina; Tomasi, Aldo; Vinceti, Marco; Ponti, Giovanni
abstract

BackgroundDiameter represents a controversial parameter in the diagnosis of melanoma (MM). The ABCD rule considers it an important diagnostic parameter, while some authors decrease its relevance. Also, its measurement is not always reliable, histological methods being usually used. The current study aims to compare histological and digital measurements and to evaluate the correlation between MM head- and trunk diameter and thickness. Methods The study population was subdivided according to diameter subgroups considering head- and trunk-limbs lesions separately. Digital diameters measured by an automatic software referring to 477 MM images were compared to diameters reported on the pathologist’s records. Clinical and histological information was also considered, and the correlation between diameter and thickness was assessed. Odds ratios (OR) were computed for different diameter subgroups. Results Mean digital diameters of head MMs and trunk limbs MMs exceeded histological measurements by 11% and 20%, respectively. In head MMs, no correlation between diameter and thickness was observable, whereas in the trunk-limbs group a direct relationship between thickness and diameter was noticed. ORs for non in situ vs in situ andfor ≥ 1 mm vs < 1 mm thick were low for small lesions, increasing for larger ones, indicating that the latter are more likely to be thick. ConclusionMM diameter should be assessed digitally to avoid tissue shrinkage after biopsy and imprecise in vivo measurements. Although nearly 10% of MMs might escape an early diagnosis based only on the D of the ABCD rule, MM diameter may be related to its thickness.The evaluation with a computer vision system should be recommended for small pigmented lesions (<6mm) in order to reduce the percentage of misdiagnosed smaller MMs and to better evaluate the parameter E of the ABCDE rule (Evolving lesion).

2013 - Sphenoid asymmetry associated to other skeletal anomalies in a clear cut case of PTCH1 mutated Gorlin-Goltz syndrome: a novel finding? [Poster]
Ruini, Cristel; Pastorino, Lorenza; Malagoli, Marzio; Bruno, Walter; Ghiorzo, Paola; BIANCHI-SCARRÀ, Giovanna; Loschi, Pietro; Pellacani, Giovanni; Tomasi, Aldo; Farnetani, Francesca; Mandel, Victor Desmond; Ponti, Giovanni
abstract

Gorlin-Goltz syndrome is an autosomal dominantly inherited disorder linked to PTCH1 mutation, recognized by a collection of clinical and radiologic signs (macrocephaly, frontal bossing, multiple intracranial calcifications including falx cerebri and atlanto-occipital ligament). We describe here a the case of a family with clear cut criteria for Gorlin-Goltz syndrome presenting the association of cranio-facial and skeletal anomalies together with a peculiar sphenoid variant. Two patients, father and son, were examined because of multiple basal cell carcinomas and keratocystic odontogenic tumours. Other suggestive findings were multiple positive family history, typical skeletal anomalies and a novel PTCH1 germline mutation (c.1041delAA). Craniofacial and other skeletal anomalies displayed at 3D and helical CT scan were: macrocephaly, skull base asymmetry (positional plagiocephaly), mandibular prognathism, mandibular condylar deformation with hyperplasia of the coronoid process, bifidity of multiple ribs and giant multilocular odontogenic jaw cysts. Extensive multilamellar calcifications were found in falx cerebri, tentorium, falx cerebelli and in the apical segment of the atlanto-occipital ligament. Thoracic anomalies included bifid left 3rd, 4th, 5th and 6th rib, dismorphic body of the 3rd thoracic vertebra, dorsolumbar scoliosis, sacrum acutum. Interestingly, 3D-CT scan showed asymmetry of both sphenoid wings with thickening of the left wing sphenoid wing together with irregularity of the architecture of trabecular bone with alternating osteolytic and sclerotic areas. Abnormalities of the sphenoid bone are not very common, and consist of differently aggressive entities: some of them are typical of the pediatric age in few hereditary and congenital disorders. The application of new criteria (i.e. peculiar calcifications of ligaments and sphenoid asymmetry) to a wider case series can lead to the early diagnosis of Gorlin syndrome, especially in pediatric patients, when the full phenotype is not yet expressed. The inclusion of bifid ribs as a novel major criteria and the recognition of peculiar cranial anomalies such as sphenoid asymmetry, well detected at volume CT reconstruction, might be useful for the recognition and characterization of misdiagnosed cases.

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 somatic affairs of BRAF: tailored therapies for advanced malignant melanoma and orphan non-V600E (V600R-M) mutations. [Articolo su rivista]
Ponti, Giovanni; Pellacani, Giovanni; Tomasi, Aldo; Gelsomino, F.; Spallanzani, A.; Depenni, R; Al Jalbout, S.; Simi, L.; Garagnani, L.; Borsari, S.; Conti, A.; Ruini, C.; Fontana, A.; Luppi, G.
abstract

BRAF V600R-M-D are uncommon mutations, not included in the experimental protocols of BRAF selective inhibitors. We report the evaluation of correlations among different types of BRAF somatic mutations in melanoma and their management with BRAF inhibitors. 21 patients with BRAF mutated metastatic melanoma were enrolled in the protocol with BRAF inhibitors for compassionate use at the University of Modena. Hot spot V600E mutations were found in 19 patients. V600R mutation and double (V600E -V600M) mutation were identified in two melanomas. In one case, V600K mutation was found. Two screening failures were noted. Mean progression free survival at follow-up of to 8 weeks, was 7.6 months. Five patients had a very short follow-up and the experimental protocol is still ongoing, so we cannot provide complete follow-up data. However, all of them are still under treatment and disease progression free. An objective response with few side effects was observed in all patients. in vitro studies with the aim of testing drug sensitivity.

2012 - Ameloblastoma: a neglected criterion for nevoid basal cell carcinoma (Gorlin) syndrome. [Articolo su rivista]
Ponti, Giovanni; L., Pastorino; A., Pollio; S., Nasti; Pellacani, Giovanni; M. D., Mignogna; Tomasi, Aldo; C. D., Forno; Longo, Caterina; G., Bianchi Scarrà; G., Ficarra; Seidenari, Stefania
abstract

Ameloblastomas are considered to be aggressive and locally invasive neoplasms derived from odontogenic epithelium with a tendency for recurrence and bone destruction. Although the relationship between nevoid basal cell carcinoma syndrome (NBCCS) and ameloblastoma is less frequent, it might constitute a peculiar stigmata of this hereditary disorder. The objective of the current study was to evaluate whether a combined clinical and biomolecular approach could be useful for the identification of NBCCS among patients with a diagnosis of ameloblastoma. The authors collected ameloblastoma tumors recorded in the databases of the Pathology Departments of the University of Modena during the period 1991-2011. Family trees were drawn for all 41 patients affected by these specific odontogenic tumors. Two patients with ameloblastoma were also affected by multiple basal cell carcinomas and odontogenic keratocysts tumors (OKCTs) achieving the requested clinical criteria for the diagnosis of NBCCS. The clinical diagnoses were confirmed by the identification of two different novel PTCH1 germline mutations (c.2186A > T [p.K729 M]; c.931insA) in those unrelated patients. Clinical ameloblastoma findings can be used as screening for the identification of families at risk of NBCCS. Ameloblastomas diagnosis warrants the search for associated cutaneous basal cell carcinomas and other benign and malignant tumors related to NBCCS. Thus, we propose the inclusion of ameloblasoma as criterion for the identification of NBCCS.

2012 - Diagnostic and pathogenetic role of café-au-lait macules in nevoid basal cell carcinoma syndrome [Articolo su rivista]
Ponti, Giovanni; Tomasi, Aldo; Pastorino, Lorenza; Ruini, Cristel; Guarneri, Carmelo; Mandel, Victor Desmond; Seidenari, Stefania; Pellacani, Giovanni
abstract

Café au lait spots (CALS) are common dermatologic findings that can at the same time arise in a variety of pathologic conditions such as Neurofibromatosis type 1 (NF1), together with numerous hereditary syndromes for which they represent either diagnostic criteria or associated elements (McCune Albright, Silver-Russell, LEOPARD, Ataxia-Telangiectasia). A review of the literature also revealed two cases of association with NBCCS. We report here the case of a female proband with CALS associated to Nevoid Basal Cell Carcinoma Syndrome (NBCCS) with known PTCH1 germline mutation (C.1348-2A&gt;G) who had been misdiagnosed with NF1 in her childhood because of 5 CALS and cutaneous nodules. The patient presented a giant cell tumor of the skin, palmar and calcaneal epidermoidal cystic nodules, odontogenic keratocystic tumors and deformity of the jaw profile. Her family history brought both her brother and father to our attention because of the presence of KCOTs diagnosed at early age: after genetic testing, the same PTCH1 germline mutation was identified in the three family members. Clinical criteria are used for discerning NF1 diagnosis (size, number and onset age), while there are no definite guidelines concerning CALS except for their presence. In our experience, we have noted an association of CALS with NBCCS; this seems interesting because we already know clinical criteria are a dynamic entity and can be modified by epidemiologic evidences.

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 - Mutazioni somatiche di BRAF in pazienti affetti da melanoma maligno metastatico ed efficacia clinica degli approcci terapeutici a bersaglio molecolare con inibitori di BRAF [Poster]
Ponti, Giovanni; Pellacani, Giovanni; Simi, Lisa; Depenni, Roberta; Spallanzani, Andrea; Gelsomino, Fabio; Pollio, Annamaria; Borsari, Stefania; Fabiano, Antonella; Mandel, Victor Desmond; Tomasi, Aldo; Luppi, Gabriele
abstract

L’introduzione di terapie a bersaglio molecolare ha rivoluzionato la prognosi dei pazienti affetti da melanoma in stadio avanzato. Attualmente è possibile determinare lo status molecolare del melanoma analizzando geni quali C-KIT, BRAF ed N-RAS per la scelta di strategie terapeutiche mirate. Mutazioni di BRAF si riscontrano in circa il 50% dei melanomi: tra queste la V600E e la V600K sono maggiormente frequenti e ben studiate nei pregressi protocolli di sperimentazione fase II/III con inibitori di BRAF. Questi ultimi (Dabrafenib e Vemurafenib) hanno dimostrato chiara efficacia nell’indurre risposte obiettive nel melanoma avanzato. Con il presente lavoro presentiamo le preliminari valutazioni delle correlazioni tra tipologie di mutazioni somatiche del gene BRAF riscontrate in una coorte di melanomi in stadio IV e le risposte cliniche a tale inibitori selettivi. A partire dal giugno 2011, 19 pazienti (10 M; 9F; età media 55 anni) affetti da melanoma stage IV BRAF+ sono stati arruolati in protocolli terapeutici con Dabrafenib (150 mg 2 volte/die) o Vemurafenib (960 mg 2volte/die) presso l’Università di Modena. Il restaging strumentale della malattia veniva eseguito a 8 settimane dall’inizio della terapia. Sono state riscontrate mutazione hot spot V600E (c.1799T&gt;A) in 18 pazienti, in due differenti pazienti sono state individuate le rare mutazioni V600M (c.1798G&gt;A) e V600R (c.1790T&gt;G). In un melanoma è stata evidenziata una doppia mutazione (V600E; V600M) ed il relativo paziente, trattato con Dabrafenib, ha presentato una rapida regressione delle importati lesioni metastatiche. In due casi la ricerca mutazionale in BRAF, risultata inizialmente negativa, è stata poi riscontrata in una successiva analisi. I dati clinici preliminari evidenziano una risposta obiettiva già nelle primissime settimane di terapia, buona tolleranza con scarsi effetti collaterali sia nei pazienti con mutazioni V600E che in quelli con le rare mutazioni V600 M e V600R. Il periodo di sopravvivenza medio libero da progressione dei 15 pazienti con follow-up di almeno 8 settimane è stato di 7 mesi. In 9 pazienti persiste la risposta terapeutica ad oggi. La doppia mutazione di BRAF potrebbe costituire un fattore prognostico positivo per la risposta agli inibitori di BRAF. Nei casi negativi alla ricerca mutazionale, dovrebbe essere routinariamente impiegate indagini più approfondite atte ad evidenziare non solo la presenza della comune V600E ma anche delle varianti meno comuni degli esoni 11 e 15.

2012 - Neoplasie maligne e benigne associate al melanoma multiplo: coinvolgimento di MITF, PTEN and CDKN2A nella cancerogenesi melanocitaria multipla. [Poster]
A., Pollio; S., Seidenari; Pellacani, Giovanni; M., Rodolfo; S., Frigerio; A., Maurichi; D., Turchetti; A., Elmakky; L., Pastorino; M. D., Mignogna; Tomasi, Aldo; Ponti, Giovanni
abstract

Background: Il fenotipo melanomi multipli è un interessante modello di ricerca per lo studio e l’identificazione di mutazioni germinali che sottendono alla maggiore suscettibilità neoplastica cutanea e viscerale, individuale e familiare. L’analisi della storia clinica di gruppo di paziente affetti da melanoma multiplo ha evidenziato una non trascurabile incidenza di neoplasie polidistrettuali associate al fenotipo melanoma multiplo. Materiali e metodi: Lo studio ha analizzato in modo retrospettivo la presenza di neoplasie benigne e maligne in un gruppo di 49 pazienti con diagnosi di melanoma multiplo rapportate ad un gruppo controllo di 49 pazienti con diagnosi di melanoma singolo selezionato per medesima età, sesso e durata del follow-up. I test genetici mutazionali sono stati eseguiti in tutti i pazienti il cui quadro clinico dava adito al sospetto di una mutazione germinale sottesa. Risultati: Una prevalenza statisticamente signficativa (P<0.0001) di neoplasie benigne e maligne è stata evidenziata nel gruppo di pazienti affetti da melanoma multiplo rispetto al gruppo controllo. Le neoplasie maligne riscontrate sono state 27 di cui la più frequente è stata il carcinoma basocellulare cutaneo (n=10, 37.1%), seguito dall’adenocarcinoma del colon-retto (n=4, 14.8%), dall’adenocarcinoma della prostata (n=3, 11.1%), dal carcinoma mammario (n=2, 7.4%), dal carcinoma papillifero della tiroide (n=2, 7.4%), dall adenocarcinoma pancreatico (n=2, 7.4%), dal carcinoma renale (n=2, 7,4%), dal carcinoma orale (n=1, 3.7%), dall’adenocarcinoma del fegato(n=1, 3.7%), dal linfoma a cellule B (n=1, 3.7%), dal mieloma multiplo (n=1, 3.7%), dal carcinoma vescicale (n=1, 3.7%) ed infine dal carcinoma dello stomaco (n=1, 3.7%). Nel gruppo controllo non è stata riportata alcuna neoplasia maligna associata. Dieci neoplasie benigne sono state riportate a carico di più organi tra i pazienti con melanoma multiplo. I test genetici hanno svelato mutazioni germinali a carico di geni quali PTEN, MITF E318K, CDKN2A, MC1R. In tre pazienti con melanomi multipli e storia personale e/o individuale di neoplasie renali a cellule chiare è stata rilevata coesistenza di mutazioni germinali di CDKN2A, MC1R e MITF . Conclusioni: Una sorveglianza oncologica accurata è raccomandabile nei pazienti affetti da melanoma multiplo. È compito del clinico selezionare in base alla storia clinica riportata i pazienti da candidare ai test genetici per un ulteriore approfondimento diagnostico. Infatti il managment clinico del paziente può essere direttamente influenzato dal tipo di mutazione diagnosticata sia per quanto concerne la terapia che la pianificazione del follow-up.

2012 - Novel PTCH1 Mutations in Patients with Keratocystic Odontogenic Tumors Screened for Nevoid Basal Cell Carcinoma (NBCC) Syndrome. [Articolo su rivista]
L., Pastorino; A., Pollio; PELLACANI, Giovanni; C., Guarneri; P., Ghiorzo; LONGO, Caterina; W., Bruno; F., Giusti; S., Bassoli; G., Bianchi Scarrà; C., Ruini; S., Seidenari; TOMASI, Aldo; PONTI, Giovanni
abstract

Keratocystic odontogenic tumors (KCOTs) are cystic tumors that arise sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). NBCCS is a rare autosomal dominantly inherited disease mainly characterized by multiple basal cell carcinomas, KCOTs of the jaws and a variety of other tumors. PTCH1 mutation can be found both in sporadic or NBCCS associated KCOTs. The aim of the current study was to assess whether a combined clinical and bio-molecular approach could be suitable for the detection of NBCCS among patients with a diagnosis of keratocystic odontogenic tumors (KCOTs). The authors collected keratocystic odontogenic tumors recorded in the database of the Pathology Department of the University of Modena and Reggio Emilia during the period 1991-2011. Through interviews and examinations, family pedigrees were drawn for all patients affected by these odontogenic lesions. We found out that 18 of the 70 patients with KCOTs and/or multiple basal cell carcinomas actually met the clinical criteria for the diagnosis of NBCCS. A wide inter- and intra-familial phenotypic variability was evident in the families. Ameloblastomas (AMLs) were reported in two probands that are also carriers of the PCTH1 germline mutations. Nine germline mutations in the PTCH1 gene, 5 of them novel, were evident in 14 tested probands. The clinical evaluation of the keratocystic odontogenic tumors can be used as screening for the detection of families at risk of NBCCS. Keratocystic odontogenic lesions are uncommon, and their discovery deserves the search for associated cutaneous basal cell carcinomas and other benign and malignant tumors related to NBCCS.

2012 - Overwhelming response to Dabrafenib in a patient with double BRAF mutation (V600E; V600M) metastatic malignant melanoma [Articolo su rivista]
Ponti, Giovanni; Tomasi, Aldo; Pellacani, Giovanni
abstract

ABSTRACT: The recent findings brought the necessity of testing the mutational status of a series of genes which had been already identified as responsible for melanomas development and progression, such as BRAF, CKIT and PTEN: the consequent results are, in fact, essential to guide the assessment of the novel treatment protocols based on tailored targeted therapies. We present here the case of a 66 year-old male patient, diagnosed with an advanced melanoma in June 2011, and treated with Dabrafenib for double mutant metastatic disease. The patient was referred to our attention for a large exophytic malignant melanoma on the left shoulder. After complete surgical excision and elective lymph node dissection for presence of metastatic sentinel lymph node, the patient has started high-dose interferon alfa- 2b injections as adjuvant therapy for a complete negative staging. The treatment was interrupted in August 2011 due to the appearance of metastatic lymph nodes. Tumor burden was rapidly growing reaching in few months the size of a tennis ball for the tumor mass located in the shoulder. Mutational study of the tumor revealed a double BRAF mutation on V-600E and V600M. This finding incited us to enroll the patient in compassionate Dabrafenib clinical trial. The therapy was started on may 2012 at 150 mg bid dosage. Almost surprisingly for the rapidity of the effect, one week later the lesion on the shoulder has reduced its size by 60% and one month later it has completely disappeared from sight. CT scan of June 2012 documented the astonishing clinical response.

2012 - Patched homolog 1 gene mutation (p.G1093R) induces nevoid basal cell carcinoma syndrome and non-syndromic keratocystic odontogenic tumors: A case report. [Articolo su rivista]
Ponti, Giovanni; A., Pollio; L., Pastorino; Pellacani, Giovanni; Magnoni, Cristina; S., Nasti; G., Fortuna; Tomasi, Aldo; G. B., Scarrà; Seidenari, Stefania
abstract

Mutations in the Patched homolog 1 (PTCH1) gene lead to an autosomal dominant disorder known as nevoid basal cell carcinoma syndrome (NBCCS) or Gorlin syndrome (GS). Several PTCH1 mutations have been observed in NBCCS associated with keratocystic odontogenic tumors (KCOTs), including non-syndromic KCOTs. The missense mutation c.3277G>C (p.G1093R) in exon 19 of the PTCH1 gene has only been reported in non-syndromic KCOTs. The present study reports for the first time a familial case (father and daughter) of NBCCS and KCOTs, carrying the same c.3277G>C (p.G1093R) germline mutation. This observation suggests that this missense mutation is involved in the pathogenesis of NBCCS as well as in a subset of non-syndromic KCOTs. The identification of a missense mutation may lead to an earlier diagnosis of NBCCS.

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 - Detection of serum protein profile changes in atherosclerosis [Abstract in Rivista]
Dursun, Evrim; Ozben, Beste; Monari, Emanuela; Cuoghi, Aurora; Bergamini, Stefania; Tomasi, Aldo; Ozben, Tomris
abstract

nd

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 - Role of proteomics to differentiate between benign and potentially malignant pancreatic cysts [Articolo su rivista]
Cuoghi, Aurora; Farina, A; Z'Graggen, K; Dumonceau, Jm; Tomasi, Aldo; Hochstrasser, Df; Genevay, M; Lescuyer, P; Frossard, J. L.
abstract

Pancreatic cystic neoplasms represent 10-15% of primary cystic masses of the pancreas. While pancreatic cysts are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice, the diagnosis of pancreatic cystic neoplasms remains unsatisfactory because available diagnostic techniques proved not sensitive enough so far. This study was designed to characterize the proteomic pattern of pancreatic cyst fluids obtained from various cystic lesions. Cyst fluids were collected by direct puncture during open surgery to avoid any possible contamination from other tissues. CEA, CA-19-9 and amylase concentrations were measured using specific immunoassays. After immunodepletion and fractionation by SDS-PAGE, proteins were digested and analyzed by LC-MS/MS. Specific histological lesions were found to be associated with distinct protein patterns. Interestingly, some of these proteins have been proposed as biomarkers of pancreatic cancer. Immunoblots allowed verifying the differential expression in specific cyst fluids of two selected proteins, olfactomedin-4 and mucin-18. Finally, immunohistochemistry was performed to correlate these data with the expression pattern of olfactomedin-4 and mucin-18 in pancreatic cyst tissues. Results from this study indicate that proteomic analysis of cyst fluid could provide reliable candidates for developing new biomarkers for the preoperative management of malignant and premalignant pancreatic cysts.

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

2010 - Proteomic profiling during atherosclerosis progression using SELDI-TOF-MS: Effect of darbepoetin treatment [Articolo su rivista]
Dursun, E; Monari, Emanuela; Cuoghi, Aurora; Bergamini, Stefania; Ozben, B; Suleymanlar, G; Tomasi, Aldo; Ozben, T.
abstract

Narrowing of the arteries due to atherosclerosis may lead to congestive heart failure (CHF). It is advantageous to perform atherosclerosis studies in apolipoprotein E-deficient (Apo E(-/-)) mice models, which develop atherosclerosis very rapidly in comparison to humans. Darbepoetin is a synthetic erythropoietin analogue and stimulates erythropoiesis. The aim of this study was to explore the effect of 16 weeks of darbepoetin treatment on serum protein profiles in Apo E(-/-) mice during atherosclerosis progression. Serum proteomic analyses were performed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) in the darbepoetin-treated and non-treated (control) Apo E(-/-) mice groups. The protein profiles obtained using three different chips, CM-10 (weak cation exchange), H50 (reversed-phase) and IMAC-30 (immobilized metal affinity capture), were statistically analyzed using the ProteinChip data manager 3.0 program. At the end of 16 weeks of darbepoetin treatment, there was no significant difference in the size and degree of atherosclerotic lesions between the darbepoetin and control mice groups. In contrast, 145 protein/peptide-clustering peaks, >5kDa, had statistically significant differences in their peak intensities between the darbepoetin and control mice groups (p<0.05). That the proteomic profiles of darbepoetin-treated Apo E(-/-) mice were found to differ from those of the control group indicates a potential beneficial role of darbepoetin in atherosclerosis. Our study contributes to understanding the effects of darbepoetin on protein/peptide expressions during atherosclerosis development.

2010 - Proteomic profiling in apolipoprotein E-deficient mice during atherosclerosis progression [Articolo su rivista]
E., Dursun; B., Ozben; Monari, Emanuela; Cuoghi, Aurora; Tomasi, Aldo; T., Ozben
abstract

Atherosclerosis and related complications are a major worldwide cause of human morbidity and mortality. It is advantageous to perform atherosclerosis studies in the apolipoprotein E-deficient (Apo E−/−) mouse model, which develops atherosclerosis very fast in comparison to humans. The aim of this study was to compare serum protein profiles in Apo E−/− mice during atherosclerosis progression with the data of control C57BL/6 mice. Serum proteomic analyses were performed using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). The protein profiles obtained using three different chips, CM-10 (weak cation exchange), H50 (reversed-phase) and IMAC-30 (immobilized metal affinity capture) were statistically analyzed using the ProteinChip data manager 3.0 program. At 20 weeks of age, all Apo E−/− mice developed early atherosclerotic lesions. The peak intensities of 742 serum protein/peptide clusters were found to be different between the atherosclerotic and control mice groups, and the differences reached statistical significance for 107 serum protein/peptide clusters (p<0.05). This study contributes to understanding the changes in serum protein/peptide profiles during atherosclerosis development and may inform discovery of new protein biomarkers for early diagnosis of atherosclerosis.

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 - Proteomic profiling during atherosclerosis progression: Effect of nebivolol treatment [Articolo su rivista]
Ozben, B; Dursun, E; Monari, Emanuela; Cuoghi, Aurora; Bergamini, Stefania; Tomasi, Aldo; Ozben, T.
abstract

There is a great need for the identification of biomarkers for the early diagnosis of atherosclerosis and the agents to prevent its progression. The aim of this study was to explore the effect of 24 week of nebivolol (a third-generation vasodilatory beta-blocker) treatment on serum protein profiles in Apo E(-/-) mice during atherosclerosis progression. Nebivolol treated and non-treated (the control group) groups consisted of 10 genetically modifiedhomozygous Apo E(-/-) mice. Proteomic analyses were performed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the serum samples from the nebivolol treated and non-treated Apo E(-/-) mice. The protein profiles obtained using three different chips, CM10 (weak cation-exchange), H50 (reverse phase), and IMAC30-Cu(2+) (immobilized metal affinity capture) were statistically analyzed using the ProteinChip data manager 3.0 program. At the end of 24 week of nebivolol-treatment period, a total of 662 protein/peptide clustering peaks were detected using 12 different conditions and reading with high and low intensity laser energy. The highest total number of protein/peptide clusters was found on H50 chip array. The peak intensities of 95 of the 662 protein/peptide clusters were significantly different in the nebivolol-treated atherosclerotic group in comparison to the non-treated control mice groups (P < 0.05). Forty-three protein/peptides were up-regulated (high signal intensity) while 52 protein/peptides had lower signal intensity (down-regulated) in the nebivolol-treated atherosclerotic group. The proteomic profiles of nebivolol-treated Apo E(-/-) mice were different than the control group indicating a potential role of nebivolol in atherosclerosis. Our study contributes to understand the efficacy of nebivolol on serum protein/peptide profiles during atherosclerosis development.

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 - Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage [Articolo su rivista]
Giuliani, Daniela; C., Mioni; Bazzani, Carla; Zaffe, Davide; A. R., Botticelli; S., Capolongo; A., Sabba; M., Galantucci; Iannone, Anna; P., Grieco; E., Novellino; G., Colombo; Tomasi, Aldo; A., Catania; Guarini, Salvatore
abstract

Background and purpose: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. Experimental approach: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP-alpha-MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg(-1) i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

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 - Antioxidant effect of FeAOX-6 on free radical species produced during iron catalyzed breakdown of tert-butyl hydroperoxide [Articolo su rivista]
Rota, Cristina; Tomasi, Aldo; Palozza, P.; Manfredini, S.; Iannone, Anna
abstract

There is a body of evidences demonstrating, in biological systems, a cooperative interaction between tocopherols and carotenoids. FeAOX-6 is a novel antioxidant that combines the chroman head of alpha-tocopherol and a fragment of the isoprenyl chain of lycopene. We have tested its antioxidant effect on different radical species generated in a chemical system, where peroxyl, alkoxyl and methyl radicals are generated by the ferrous ion-mediated decomposition of tert-butyl hydroperoxide. We found that FeAOX-6 has the same effectiveness of alpha-tocopherol in quenching peroxyl radical with no contribution by lycopene. The antioxidant activity of FeAOX-6 on alkoxyl and methyl radicals is comparable to that of the equimolar mixture of the parent compounds. Lycopene is able to quench alkoxyl radical, while it has no effect on peroxyl radical, showing a different antioxidant activity compared to other carotenoids, such as beta-carotene and lutein.

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 - Detection of a stable nitroxide during chemical depolymerization of heparin [Articolo su rivista]
Rota, Cristina; L., Liverani; F., Spelta; G., Mascellani; Tomasi, Aldo; Iannone, Anna
abstract

The use of low-molecular-weight heparins (LMWHs) has become common, since compared to unfractionated heparin (UFH), they have a much longer plasma half-life and lower incidence of side effects. LMWHs are derived from the depolymerization of UFH, obtained either chemically, physically or enzymatically. We employed electron spin resonance (ESR) spectroscopy to study the depolymerization of UFH by copper in the presence of hydrogen peroxide. A stable nitroxide radical was detected. This could be generated by the hydroxyl radical attack either to the N-SO3- group or to free amino groups present in the UFH preparation.

2006 - Il Progetto della Facoltà di Medicina e Chirurgia della Università di Modena e Reggio Emilia "Le Cure Primarie nel CDL in Medicina e Chirurgia" [Articolo su rivista]
Tomasi, Aldo; Becchi, Maria Angela
abstract

Viene descritto il Progetto Didattico realizzato nel CDL in Medicina e Chirurgia della Università di Modena e Reggio Emilia allo scopo di sviluppare una formazione di base nell'area delle Cure Primarie, come auspicato dal DM 28.11.2000. Sono descritte le caratteristiche generali del Corso in termini di finalità, obiettivi formativi, modalità didattiche, docenti, setting formativi e aspetti organizzativi. Viene focalizzata l'attenzione sul carattere di "Corso Integrato" che il Progetto viene ad assumere, essendo basato sulla collaborazione fra Docenti Universitari e Professionisti di Azienda USL, compresi i medici di medicina generale. Viene focalizzata l'attenzione anche sulla "Didattica teorico-pratica" basata su seminari integrati fra Docenti Universitari e Professionisti aziendali e didattica tutoriale negli ambulatori dei mmg e in altri setting delle CP.

2006 - La medicina universitaria nello sviluppo delle cure primarie [Articolo su rivista]
Becchi, Maria Angela; P., Paolucci; G., Salvioli; Tomasi, Aldo; N., Carulli
abstract

Sono riconosciute le lacune di formazione nell'area delle Cure Primarie (CP) attualmente presenti in Italia nonostante le normative vigenti prevedano specifici percorsi nel pre-laurea (DM 28.11.00 All. 46 ) e nel post-laurea (DM 1.8.2005). Queste lacune hanno avuto nel tempo inevitabili ricadute sulle scelte professionali dei medici, oggi prevalentemente specialistiche in ambito ospedaliero.

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 - Role of oxidative stress in progressive kidney failure [Abstract in Rivista]
Tomasi, Aldo; S., Uggeri; Bergamini, Stefania; DELLA CASA, Lara; Albertazzi, Alberto; L., Lucchi; Iannone, Anna
abstract

not available

2006 - The oxidation of 2 ',7 '-dichlorofluorescin to reactive oxygen species: A self-fulfilling prophesy? [Articolo su rivista]
M. G., Bonini; Rota, Cristina; Tomasi, Aldo; R. P., Mason
abstract

The oxidation of 2',7'-dichlorofluorescin (DCFH) and its diacetate form (DCFHDA) by the HRP/peroxynitrite system was investigated. Both DCFH and DCFHDA were oxidized to fluorescent products. A major anomaly, however, was the observation that fluorescence continued to build Lip long after peroxynitrite total decomposition and the initial HRP compound I reduction, suggesting the production of oxidants by the system. Indeed, preformed HRP compound I was instantly reduced by DCFH and DCFHDA to compound II with the obligate formation of DCF center dot- semiquinone and DCFHDA-derived radicals. Catalase strongly inhibited fluorescence and EPR signals, suggesting the intermediate formation of H2O2. Taken together the data indicate that peroxynitrite rapidly oxidizes HRP to HRP compound I, which is reduced by DCFH and its diacetate form with the concomitant formation of DCF center dot- semiquinone and DCFH DA-derived radicals. These are oxidized by O-2, producing O-2(center dot-) - (as demonstrated by EPR and oxygen consumption experiments), which dismutates to produce H2O2, which serves to fuel further DCFH/DCFHDA oxidation via HRP catalysis. Also DCFHDA was shown to be considerably more resistant to oxidation than its hydrolyzed product DCFH, presumably because of the absence of the easily oxidizable phenol moieties. DCFHDA/DCFH have been used to study free radical production in a variety of systems. Our findings demonstrate that this assay is subject to a serious artifact in that it produces what it is purported to measure; therefore, its use in biological systems should be approached with caution. Published by Elsevier Inc.

2006 - α-Tocopherol amplifies benzoyl peroxide free radical decomposition in a chemical system [Articolo su rivista]
Rota, Cristina; Tomasi, Aldo; Iannone, Anna
abstract

Benzoyl peroxide is commonly used in the treatment of acne, even though some adverse effects have been reported, probably mediated by the formation of peroxide-derived free radicals and the depletion of antioxidants. In the present work we have studied, in a chemical system, the effect of a-tocopherol on benzoyl peroxide radical decomposition to analyse the presence of an interaction between these two compounds, leading to an enhanced peroxide-cytotoxicity, as we have previously reported. Under our experimental conditions alpha-tocopherol strongly amplified the peroxide free radical decomposition occurring either in the presence or in the absence of UV irradiation, and lead to the formation of an unknown radical species in addition to benzoyloxy, phenyl and tocopheroxyl free radicals. The results of this study show that the enhancement of benzoyl peroxide toxicity in cells exposed simultaneously to this peroxide and alpha-tocopherol, is likely due to the generation of the detected radical species.

2005 - Activation of an efferent cholinergic pathway produces strong protection against myocardial ischemia/reperfusion injury in rats [Articolo su rivista]
Mioni, C.; Bazzani, Carla; Giuliani, Daniela; Altavilla, D.; Leone, S.; Ferrari, Anna; Minutoli, L.; Bitto, A.; Marini, H.; Zaffe, Davide; Botticelli, Ar; Iannone, Anna; Tomasi, Aldo; Bigiani, Albertino; Bertolini, Alfio; Squadrito, F.; Guarini, Salvatore
abstract

Objective: A vagus nerve-mediated, brain cholinergic protective mechanism activated by melanocortin peptides is operative in conditions of circulatory shock; moreover, there is anatomical evidence of dual vagal-cardiac efferent pathways in rats, which could play different roles in controlling heart function. Therefore, we investigated the role and functional mechanism of such vagal efferent pathway(s) in an experimental model of ischemic heart disease. Design: Randomized experimental study. Setting: Research laboratory. Subjects: Adult Wistar rats of either sex. Interventions: After bilateral cervical vagotomy (with or without pretreatment with atropine), efferent vagal fibers were electrically stimulated in rats subjected to coronary artery occlusion (5 mins) followed by reperfusion (5 mins). Other rats (intact, vagotomized, or pretreated with atropine) were treated with nanomolar doses of melanocortin peptides. Measurements and Main Results: Electrical stimulation of efferent vagal fibers (5 V, 2 msecs,1-9 Hz, for the whole period of ischemia/reperfusion) strongly reduced the high incidence of severe arrhythmias and lethality, reduced the increase in free radical blood levels and left-ventricle histologic alterations, and augmented the extracellular signal-regulated kinase activation. Treatment with the melanocortin peptides adrenocorticotropin and gamma(2)-melanocyte-stimulating hormone (162 nmol/kg intravenously or 16.2 nmol/kg intracerebroventricularly, during coronary occlusion) produced the same protective effects of electrical stimulation and with the same muscarinic acetylcholine receptor-dependent mechanism, seemingly through brain activation (mediated by melanocortin MC3 receptors, as previously described) of such efferent vagal pathway. Conclusions: The present results give evidence for the identification of a protective, melanocortin-activated, efferent vagal cholinergic pathway, operative in conditions of myocardial ischemia/reperfusion. These data suggest that melanocortins and pertinent compounds able to activate such a pathway could provide the potential for development of a new class of drugs for a novel approach to management of ischemic heart disease.

2005 - Application of electron paramagnetic resonance spectroscopy for detection of nitric oxide and free iron in mitochondria. [Abstract in Rivista]
Av, Kozlov; S., Bahrami; H., Nohl; H., Redl; Tomasi, Aldo
abstract

not available

2005 - Combination of iron overload plus ethanol and ischemia alone give rise to the same endogenous free iron pool [Articolo su rivista]
O., Sergent; Tomasi, Aldo; Ceccarelli, Daniela; Masini, Alberto; H., Nohl; P., Cillard; J., Cillard; Ya, Vladimirov; Av, Kozlov
abstract

Iron overload aggravates tissue damage caused by ischemia and ethanol intoxication. The underlying mechanisms of this phenomenon are not yet clear. To clarify these mechanisms we followed free iron (loosely bound redox-active iron) concentration in livers from rats subjected to experimental iron overload, acute ethanol intoxication, and ex vivo warm ischemia. The levels of free iron in non-homogenized liver tissues, liver homogenates, and hepatocyte cultures were analyzed by means of EPR spectroscopy. Ischemia gradually increased the levels of endogenous free iron in liver tissues and in liver homogenates. The increase was accompanied by the accumulation of lipid peroxidation products. Iron overload alone, known to increase significantly the total tissue iron, did not affect either free iron levels or lipid peroxidation. Homogenization of iron-loaded livers, however, resulted in the release of a significant portion of free iron from endogenous depositories. Acute ethanol intoxication increased free iron levels in liver tissue and diminished the portion of free iron releasing during homogenization. Similarly to liver tissue, the primary hepatocyte culture loaded with iron in vitro released significantly more free iron during homogenization compared to non iron-loaded hepatocyte culture. Analyzing three possible sources of free iron release under these experimental conditions in liver cells, namely ferritin, intracellular transferrin-receptor complex and heme oxygenase, we suggest that redox active free iron is released from ferritin under ischemic conditions whereas ethanol and homogenization facilitate the release of iron from endosomes containing transferrin-receptor complexes.

2005 - Comparison between hydroperoxides and malondialdehyde as markers of acute oxidative injury during hemodialysis [Articolo su rivista]
L., Lucchi; Iannone, Anna; Bergamini, Stefania; L., Stipo; S., Perrone; S., Uggeri; V., Gatti; F., Ferrari; Tomasi, Aldo; Albertazzi, Alberto
abstract

An increased free-radical production has been documented during hemodialysis (HD) particularly when bio-incompatible membranes are utilized. These highly reactive free radicals can cause damage through several pathways, one of the best known being lipid peroxidation. Malondialdehyde (MDA) is a product of lipid peroxidation, which can partly be removed by HD due to its low molecular weight and water solubility. Hydroperoxides are predominantly found in lipid substances, and therefore their removal by HD could be difficult. We evaluated the behavior of these two by-products of lipid peroxidation during HD, comparing their behavior in three different membranes, in order to study their reliability as markers of acute oxidative injury. Fifteen stable HD patients were dialyzed with each of the following membranes: cuprophan, polyamide, and polysulfone, three sessions for every membrane. MDA and hydroperoxides were measured pre-HD and then both from the arterial and venous line at 8, 15, 30, and 240 min. During HD with cuprophan membrane MDA decreased significantly in the venous line compared with the arterial line at 8, 15, and 30 min (P < 0.05). At the end of HD, MDA was significantly reduced compared with MDA pre-HD (P < 0.05). Plasma hydroperoxides increased significantly in the venous line compared with the arterial line at 8, 15, 30, and 240 min (P < 0.05). At the end of HD, hydroperoxides had increased significantly as compared with pre-HD (P < 0.05). When the polyamide and polysulfone membranes were used, the behavior of MDA was similar to that found with cuprophan. Hydroperoxides were unchanged during HD using both membranes. MDA is not a reliable marker of acute oxidative injury during HD as it is removed during HD. Hydroperoxide measurement is a better marker of acute oxidative injury during HD.

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 - Erythrocyte susceptibility to oxidative stress in chronic renal failure patients under different substitutive treatments [Articolo su rivista]
Lucchi, L; Bergamini, Stefania; Iannone, Anna; Perrone, S; Stipo, L; Olmeda, F; Caruso, F; Tomasi, Aldo; Albertazzi, Alberto
abstract

An increased oxidative stress is now considered one of the major risk factors in chronic renal failure (CRF) patients that may be exacerbated by dialysis. It has been postulated that this increased oxidative stress might cause an augmented red blood cell (RBC) membrane lipid peroxidation with the consequent alteration in membrane deformability. The aim of this study was to evaluate RBC susceptibility to an in vitro induced oxidative stress and RBC antioxidant potential in different groups of CRF patients undergoing different substitutive treatment modalities. Fifteen end-stage CRF patients were evaluated in conservative treatment, 23 hemodialysis (HD) patients, 15 continuous ambulatory peritoneal dialysis (CAPD) patients, 15 kidney transplanted patients, and 16 controls. Their RBCs were incubated with the oxidative stress-inducing agent tert-butylhydroperoxide both in the presence and in the absence of the catalase inhibitor sodium azide, and the level of malondialdehyde (MDA) (a product of lipid peroxidation), was measured at 0, 5, 10, 15, and 30 min of incubation. In addition, the RBC content of reduced glutathione (GSH) was measured by HPLC. As opposed to the controls, RBCs from end-stage CRF patients exhibited an increased sensitivity to oxidative stress induced in vitro, both in the absence and presence of a catalase inhibitor, as demonstrated by a significantly higher level of MDA production at all the incubation times (P < 0.05). Different substitutive treatments had different impacts on this phenomenon; CAPD and kidney transplantation were able to normalize this alteration while HD was not. GSH appeared to be related to the increase in RBC susceptibility to oxidative stress; its content being significantly elevated in end-stage CRF and HD patients as compared with CAPD and transplanted patients and controls (P < 0.05). No significant changes were observed in the RBC glutathione content during the HD session. The increase of GSH in RBCs of end-stage CRF and HD patients seems to indicate the existence of an adaptive mechanism under increased oxidative stress occurring in vivo. Unlike HD, the beneficial effect of CAPD on the anemia of dialysis patients might partly be due to a condition of lower oxidative stress that might in addition counterbalance the cardiovascular negative effects of dislipidemia of CAPD patients.

2005 - Free radical generation during chemical depolymerization of heparin [Articolo su rivista]
Rota, Cristina; L., Liverani; F., Spelta; G., Mascellani; Tomasi, Aldo; Iannone, Anna; E., Vismara
abstract

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.

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 - A comparison between the antioxidant and peroxynitrite-scavenging functions of the vitamin E metabolites alpha- and gamma-carboxyethyl-6-hydroxychromans [Articolo su rivista]
F., Galli; M., Piroddi; Iannone, Anna; S., Pagliarani; Tomasi, Aldo; A., Floridi
abstract

Carboxyethyl-6-hydroxychromans (CEHC) are vitamin E metabolites with proposed in vitro antioxidant function. In this study we compared the antioxidant potency of the two main CEHC metabolites found in biological fluids (i.e., alpha-CEHC and gamma-CEHC) using two different experimental models of lipid oxidation: 1) plasma diluted 1/50 vol/vol in phosphate buffered saline (PBS) exposed to 50 muM Cu2+ ions, and 2) LDL (100 mug of proteins) exposed to different pro-oxidants as 2.5 muM Cu2+, 1 mM of the water soluble peroxyl radical generator 2,2'-Azobis(2-amidinopropane) hydrochloride (AAPH) and human macrophages (4 x 10(5) cells). Moreover, the two CEHC homologues were assessed for the inhibitory effect on the peroxynitrite (ONOO-)-induced nitration of tyrosine (Tyr). The results showed that in the concentration range 0.015-5 muM the CEHC metabolites and the hydrosoluble analogue Trolox exert similar concentration-dependent inhibition of the Cu2+-induced lipid oxidation of plasma. After in vitro exposure to tert-butyl hydroperoxide/Fe2+, CEHC formed chromanoxyl radicals with electron spin resonance spectra matching exactly those of their parent tocopherols. The LDL oxidation induced by AAPH or Cu2+ was significantly and similarly inhibited by 1 muM of both the CEHC homologues and Trolox. gamma-CEHC showed a slight but significantly higher inhibition of the macrophage-induced low-density lipoprotein (LDL) oxidation than alpha-CEHC. Both the CEHC homologues inhibit Tyr nitration induced by ONOO-. However, gamma-CEHC produced a slightly greater inhibitory effect than alpha-CEHC through the formation of the nitrated congener 5-nitro-gamma-CEHC. In all the systems under investigation, low nanomolar concentrations of CEHC (i.e., the concentration range in the blood of subjects with normal dietary intake of vitamin E) produced feeble antioxidant effects. In conclusion, gamma-CEHC and alpha-CEHC show similar concentration-dependent inhibition of plasma and LDL lipid oxidation. gamma-CEHC has a fairly higher potency than alpha-CEHC as ONOO- scavenger through the formation of 5-nitro-gamma-CEHC. CEHC metabolites show the same in vitro antioxidant chemistry of their parent tocopherols, but the characteristic hydrophilicity of these metabolites could result in different biopotency and roles. Further studies are needed to clarify whether CEHC could contribute to the antioxidant network in biological fluids and tissues.

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 - Erratum: Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors (European Journal of Pharmacology (2003) 477 (227-234)) [Articolo su rivista]
Mioni, C.; Giuliani, D.; Cainazzo, M. M.; Leone, S.; Iannone, A.; Bazzani, C.; Grieco, P.; Novellino, E.; Tomasi, A.; Bertolini, A.; Guarini, S.
abstract

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 - Red wine consumption prevents vascular oxidative stress induced by a high-fat meal in healthy volunteers [Articolo su rivista]
Ventura, Paolo; Bini, Anna; R., Panini; L., Marri; Tomasi, Aldo; Salvioli, Gianfranco
abstract

In order to investigate the effect of red wine on plasma lipid and oxidative stress parameters after a high-fat meal, fifteen healthy volunteers were studied: three days after a high-fat meal with 250 mL of water, they received the same meal with 250 mL of red wine. During both periods, serial blood samples were drawn before and 2, 4, and 8 hours after the meal to evaluate plasma lipids (cholesterol and triglycerides; retinyl palmitate), oxidative stress (D-ROM, and malondialdehyde) and antioxidant (total plasma antioxidant levels and uric acid) parameters. During the meal without wine, plasma lipid parameters increased significantly, whereas plasma total plasma antioxidant levels decreased, and a trend toward reduction of uric acid levels was seen). A similar trend in lipid parameters was observed after the meal with wine; no significant difference in individual lipid parameter trends after a meal with and without wine was observed. Wine ingestion induced higher total plasma antioxidant levels and uric acid; malondialdehyde levels remained constant after wine ingestion. Plasma D-ROM showed a significant postprandial increase in both experiments, but it was significantly lowered after wine ingestion. Our results give evidence of oxidative stress following a fat-rich meal in healthy subjects, suggesting that ingestion of red wine during a high-fat meal significantly reduces oxidative stress without inducing any significant modification in postprandial lipemia.

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 - Free Radicals, Nitric Oxide, and Inflammation: Molecular, Biochemical and Clinical Aspects [Monografia/Trattato scientifico]
Tomasi, Aldo; T., Ozben; V., Skulachev
abstract

Inflammation is the local response of a complex organism to an injury that serves as a mechanism initiating the elimination of noxious agents and of damaged tissues. It is now well understood that damaging mechanisms at the basis of very common human pathologies, such as atherosclerosis, neurodegenerative diseases, and cancer, i.e. the most common human pathologies are driven by the inflammatory process. Free radicals, and the very special free radical nitric oxide, are playing a relevant role in the pathogenesis of inflammation. The initial chapters introduce to the general knowledge necessary to understand the inflammatory process and the role played by free radical and oxidative stress. The interplay between inflammatory molecules and cell signaling is also dealt with in depth. A second part is dedicated to nitric oxide, redox regulation and antioxidant function in inflammation. The final chapters are devoted to diseases where inflammation plays the dominant role: septic shock, end-stage renal disease, neurodegenerative, ischemic and lung diseases. This book, while not covering the whole gamut of the massive literature on inflammation and human diseases, gives an updated and concise view on the major issues concerning the pivotal role of inflammation in so many different human pathologies. At the same time it gives directions for future paths of research leading to a control of the pathologic process.

2003 - Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors [Articolo su rivista]
Mioni, Chiara; Giuliani, Daniela; Mm, Cainazzo; Leone, Sheila; Iannone, Anna; Bazzani, Carla; P., Grieco; E., Novellino; Tomasi, Aldo; Bertolini, Alfio; Guarini, Salvatore
abstract

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC3 receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC3 receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC3/MC4 receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(I -24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC3 receptors, the C-terminal Arg-Phe structure being relevant.

2003 - Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria [Articolo su rivista]
Scotti, C; Iamele, L; Alessandrini, Andrea; Vannini, V; Cazzalini, O; Lazze, Mc; Melli, R; Savio, M; Pizzala, R; Stivala, La; Biglieri, S; Tomasi, Aldo; Bianchi, L.
abstract

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo. (C) 2003 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved.

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 - Redox regulation, cytokines, and nitric oxide in inflammation [Capitolo/Saggio]
Tomasi, Aldo; Bergamini, Stefania; Rota, C.; Iannone, Anna
abstract

non disponibile

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 - N-Acetylcysteine negatively modulates nitric oxide production in endotoxin-treated rats through inhibition of NF-ĸB activation [Articolo su rivista]
C., Rota; Bergamini, Stefania; F., Daneri; Tomasi, Aldo; F., Virgili; Iannone, Anna
abstract

N-Acetylcysteine (NAC) has a wide spectrum of biological activities, either related to the ability to increase intracellular thiols or directly acting as an antioxidant. We used an in vivo animal model to study NAC modulation of nitric oxide (NO) production in response to lipopolysaccharide treatment. A comparison was made between NAC and the N-[3-(aminomethyl)benzyl] acetamidine (1400W), an inhibitor of the inducible NO synthase (iNOS). Both inhibit NO production, although NAC lacks any effect if given when iNOS is already induced; this indicates that the decrease of NO generation is not due to an effect on iNOS activity. We found that the DNA binding activity of nuclear transcription factor-kappaB in peripheral blood cells was inhibited by NAC given before lipopolysaccharide, whereas tumor necrosis factor-a secretion was not affected.

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.

2001 - Melanocortin peptides prevent the ischemia and reperfusion-induced myocardial damage. [Abstract in Rivista]
Cainazzo, Maria Michela; Bazzani, Carla; Botticelli, A.; Zaffe, Davide; Tomasi, Aldo; Bini, Anna; Ferrazza, G.; Mioni, Chiara; Bertolini, Alfio; Guarini, Salvatore
abstract

Melanocortin peptides prevent the ischemia and reperfusion-induced myocardial damage.

2001 - N-Acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase [Articolo su rivista]
Bergamini, Stefania; C., Rota; R., Canali; Mg, Staffieri; F., Daneri; Bini, Anna; F., Giovannini; Tomasi, Aldo; Iannone, Anna
abstract

non disponibile

2001 - Protective effect of melanocortin peptides in rat myocardial ischemia [Articolo su rivista]
Bazzani, Carla; Guarini, Salvatore; Botticelli, A. R.; Zaffe, Davide; Tomasi, Aldo; Bini, Anna; Cainazzo, M. M.; Ferrazza, G.; Mioni, C.; Bertolini, Alfio
abstract

The influence of the melanocortin peptide ACTH-(1-24) (adrenocorticotropin) on the consequences of short-term coronary ischemia (5 min) followed by reperfusion, and the effect of the long-acting melanocortin [Nle(4),D-Phe(7)]alpha -melanocyte-stimulating hormone (NDP-MSH) on the damage induced by a permanent coronary occlusion, were investigated in anesthetized rats. Ischemia was produced by ligature of the left anterior descending coronary artery. Reperfusion-induced arrhythmias [ventricular tachycardia (VT), ventricular fibrillation (VF)] and survival rate within the 5 min following reperfusion, blood levels of free radicals detected 2 min after reperfusion by electron spin resonance spectrometry, and amount of healthy myocardial tissue, measured 72 h after permanent coronary occlusion on immunohistologically stained serial sections, were evaluated. Postischemic reperfusion induced VT in all saline-treated rats, and VF and death in a high percentage of animals (87%). In rats treated i.v. (2.5 min after coronary occlusion) with ACTH-(1-24) (0.16-0.48 mg/kg) there was a significantly dose-dependent reduction in the incidence of arrhythmias and lethality. Ischemia/reperfusion caused a large increase in free radical blood levels; treatment with ACTH-(1-24) (0.48 mg/kg i.v.) almost completely prevented this increase. In rats subjected to permanent coronary occlusion, the amount of healthy myocardial tissue was much reduced in saline-treated rats, while in rats treated s.c. with NDP-MSH (0.27 mg/kg every 12 h) it was significantly higher. The present data demonstrate, for the first time, an unforeseen property of melanocortin peptides, i.e., their ability to significantly reduce both heart ischemia/reperfusion injury and size of the ischemic area induced by permanent coronary occlusion.

2000 - Antioxidative efficacy of parallel and combined supplementation with coenzyme Q10 and d-alpha-trocopherol in mildly hypercholesterolemic subjects: a randomized placebo-controlled clinical study [Articolo su rivista]
J., Kaikkonen; K., Nyyssonen; Tomasi, Aldo; Iannone, Anna; T. P., Tuomainen; E., Porkkala Sarataho; J. T., Salonen
abstract

It has been claimed that coenzyme Q10 (Q10) would be an effective plasma antioxidant since it can regenerate plasma vitamin E. To test separate effects and interaction between Q10 and vitamin E in the change of plasma concentrations and in the antioxidative efficiency, we carried out a double-masked, double-blind clinical trial in 40 subjects with mild hypercholesterolemia undergoing statin treatment. Subjects were randomly allocated to parallel groups to receive either Q10 (200 mg daily), d-alpha-tocopherol (700 mg daily), both antioxidants or placebo for 3 months. In addition we investigated the pharmacokinetics of Q10 in a separate one-week substudy. In the group that received both antioxidants, the increase in plasma Q10 concentration was attenuated. Only vitamin E supplementation increased significantly the oxidation resistance of isolated LDL. Simultaneous Q10 supplementation did not increase this antioxidative effect of vitamin E. Q10 supplementation increased and vitamin E decreased significantly the proportion of ubiquinol of total Q10, an indication of plasma redox status in vivo. The supplementations used did not affect the redox status of plasma ascorbic acid. In conclusion, only vitamin E has antioxidative efficiency at high radical flux ex vivo. Attenuation of the proportion of plasma ubiquinol of total Q10 in the vitamin E group may represent in vivo evidence of the Q10-based regeneration of the tocopheryl radicals. In addition, Q10 might attenuate plasma lipid peroxidation in vivo, since there was an increased proportion of plasma ubiquinol of total Q10.

2000 - Ex-vivo detection of free radicals [Capitolo/Saggio]
Tomasi, Aldo; Iannone, Anna; M., Ferrari; V., Quaresima
abstract

Ex-vivo detection of free radicals

2000 - Influence of different hemodialysis membranes on red blood cell susceptibility to oxidative stress [Articolo su rivista]
L., Lucchi; Bergamini, Stefania; B., Botti; R., Rapana; A., Ciuffreda; P., Ruggiero; M., Ballestri; Tomasi, Aldo; Albertazzi, Alberto
abstract

Oxidative stress is crucial in red blood cell (RBC) damage induced by activated neutrophils in in vitro experiments. The aim of the study was to evaluate whether the bioincompatibility phenomena occurring during hemodialysis (HD) (where neutrophil activation with increased free radical production is well documented) may have detrimental effects on RBC. We evaluated RBC susceptibility to oxidative stress before and after HD in 15 patients using Cuprophan, cellulose triacetate, and polysulfone membrane. RBC were incubated with t-butyl hydroperoxide as an oxidizing agent both in the presence and in the absence of the catalase inhibitor sodium azide. The level of malonaldehyde (MDA), a product of lipid peroxidation, was measured at 0, 5, 10, 15, and 30 min of incubation. When Cuprophan membrane was used, the MDA production was significantly higher after HD, indicating an increased susceptibility to oxidative stress in comparison to pre-I-ID. The addition of sodium azide enhanced this phenomenon. Both cellulose triacetate and polysulfone membranes did not significantly influence RBC susceptibility to oxidative stress. Neither the level of RBC reduced glutathione nor the RBC glutathione redox ratio changed significantly during HD with any of the membranes used. The RBC susceptibility to oxidative stress was influenced in different ways according to the dialysis membrane used, being increased only when using the more bioincompatible membrane Cuprophan, where neutrophil activation with increased free radical production is well documented. The alterations found in this study might contribute to the reduced RBC longevity of HD patients where a bioincompatible membrane is used.

1999 - High blood levels of nitric oxide in rats subjected to prolonged respiratory arrest and their modulation during adrenocorticotropin-induced resuscitation [Articolo su rivista]
Bazzani, Carla; Bini, Anna; Mm, Cainazzo; Meletti, Eros; Tomasi, Aldo; Bertolini, Alfio; Guarini, Salvatore
abstract

Anaesthetized rats, endotracheally intubated and mechanically ventilated with room air, were subjected to a 5-min period of asphyxia by turning off the ventilator. The ventilator was then turned back on and, simultaneously, the animals were treated with either the adrenocorticotropin fragment 1-24 [ACTH-(1-24), 160 mu g/kg in a volume of 1 ml/kg i.v.] or an equivalent volume of saline. Nitric oxide (NO)-haemoglobin formation was detected ex vivo in arterial blood by electron spin resonance spectrometry; arterial blood pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for a 60-min observation period, or until prior death. During asphyxia, there was massive formation of NO (red cell concentrations 40-80 mu M), associated with a dramatic fall in mean arterial pressure and pulse pressure, marked bradycardia and ECG signs of ischaemic damage, as well as an isoelectric EEG. Treatment with ACTH-(1-24) produced a prompt (within 15 min) and long-lasting drop in NO blood levels, associated with an almost immediate (within 1 min) restoration of cardiovascular function and with a more gradual recovery of EEG, which became normal after 30-40 min; all parameters remained stable throughout the 60-min observation period. In saline-treated rats, on the other hand, there was a further increase in NO blood levels, as detected 3 min after treatment, and all died within 5-8 min. Moreover, pretreatment and treatment with S-methylisothiourea sulphate (SMT, 3 mg/kg i.v.), a relatively specific inhibitor of inducible NO synthase, inhibited NO formation, but did not affect the mortality rate (100% within 5-8 min). The present results provide the first evidence that prolonged asphyxia is associated with high blood concentrations of NO, and that the life-saving effect of melanocortin peptides in severe hypoxic conditions is associated with a complete normalization of NO blood levels. However, the lack of SMT protection in this experimental model seems to rule out the possibility that the ACTH-(1-24)-induced resuscitation is due to an effect on NO overproduction.

1999 - Nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle ischemia-reperfusion injury [Articolo su rivista]
Da, Lepore; Av, Kozlov; Ag, Stewart; Jv, Hurley; Tomasi, Aldo; Wa, Morrison
abstract

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia-reperfusion (IR) injury to rat skeletal muscle in vivo. Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion, The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitor N-nitro-L-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitor S-methylisothiourea (8 mg/kg). In contrast, nitrosoheme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg), Muscle necrosis in vitro did not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle,

1999 - Prooxidant activity of ferrioxamine in isolated rat hepatocytes and linoleic acid micelles [Articolo su rivista]
Bergamini, Stefania; C., Rota; M., Staffieri; Tomasi, Aldo; Iannone, Anna
abstract

The complex iron-desferrioxamine (ferrioxamine) is considered chemically unreactive, and not able to participate in redox cycle reactions. Desferrioxamine-dependent toxicity is, however, described in both human and animal studies. The aim of this work was to test the possibility that chelated iron, under certain circumstances, could enter redox reactions, giving an explanation of desferrioxamine side effects, Carefully prepared ferrioxamine, to obtain a 1:1 desferrioxamine:iron ratio, was added to isolated rat hepatocytes and to linoleic acid micelles. A strong prooxidant and cytotoxic effect was observed in the cells, also potentiating tert-butyl hydroperoxide-induced lipid peroxidation. In micelles, the prooxidant effect was observed only in the presence of ascorbate, which is oxidized during the process, giving rise to ascorbyl radical. Ferrioxamine, under the experimental conditions used, did not release iron, indicating that the prooxidant effect was due to iron redox cycling. The addition of desferrioxamine prevented both ferrioxamine- and tert-butyl hydroperoxide-induced lipid peroxidation and cytotoxicity. Concurrently, a nitroxide radical was detected, an indication of the radical scavenger activity of the hydroxamic moiety. No radical species was observed when ferrioxamine was added to the same system. The prooxidant effect of ferrioxamine gives a possible explanation of the reported human and animal desferrioxamine toxicity. When, in compartmentalized regions, the ratio of desferrioxamine:metal reaches 1:1, ferrioxamine is formed. In the absence of metal-free desferrioxamine, ferrioxamine can participate in redox cycling reactions, initiating lipid peroxidation and cytotoxicity.

1999 - The survival of skeletal muscle myoblasts in vitro is sensitive to a donor of nitric oxide and superoxide, SIN-1, but not to nitric oxide or peroxynitrite alone [Articolo su rivista]
Da, Lepore; Ag, Stewart; Tomasi, Aldo; Rl, Anderson; Jv, Hurley; Wa, Morrison
abstract

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2 ´(hydroxynitrosohydrazono)bis- (DETA NONOate), or to a donor of both NO and O-2, 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO-) or to DETA NONOate in the presence of an O-2(-)- generating system did not reduce their survival. The results show that NO or ONOO- alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H2O2 in this myoblast culture system.

1999 - Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage [Articolo su rivista]
A., Masini; C., Scotti; A., Calligaro; O., Cazzalini; La, Stivala; L., Bianchi; F., Giovannini; D., Ceccarelli; Muscatello, Umberto; Tomasi, Aldo; V., Vannini
abstract

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers, In the same animal models. the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a pal tial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis, In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy. (C) 1999 Elsevier Science B.V. All rights reserved.

1998 - Adrenocorticotropin counteracts the increase in free radical blood levels, detected by electron spin resonance spectrometry, in rats subjected to prolonged asphyxia [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Bini, Anna; Cainazzo, Maria Michela; Tomasi, Aldo; Bertolini, Alfio
abstract

We investigated the influence of the adrenocorticotropic fragment 1-24 [ACTH(1-24)] on the blood levels of highly-reactive free radicals in a rat model of prolonged asphyxia. Anesthetized animals were endotracheally intubated and mechanically ventilated with room air; after a 10 min stabilization period, the ventilator was turned off to induce asphyxia for 5 min; then, the ventilator was turned back on, and, simultaneously, the rats were intravenously treated with either ACTH-(1-24) (160 mu g/kg in a volume of 1 ml/kg) or equivolume saline. Free radicals were detected in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent employed (alpha-phenyl-N-tert-butylnitrone). Arterial pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for the 60 min observation period, or until prior death. At the end of the 5 min period of respiratory arrest, blood levels of free radicals were about four times higher than those of the basal, pre-asphyxia condition, arterial pressure had dramatically decreased, ECG showed marked bradycardia and signs of ischemic damage and the EEG had become isoelectric. Treatment with ACTH-(1-24) produced an immediate normalization of the blood levels of free radicals, associated with a restoration of cardiovascular function and full recovery of EEG within 30-45 min; all the saline-treated rats, on the other hand, died within 6.89 +/- 0.96 min. These results provide direct evidence that in st severe condition of prolonged asphyxia there is a rapid and massive production of highly-reactive free radicals and suggest that the resuscitating effect of adrenocorticotropin fragments in severe hypoxic conditions may be largely due to the inhibition of free radical overproduction during tissue reoxygenation.

1998 - Antioxidant activity of carotenoids: an electron spin resonance study of beta-carotene and lutein interaction with free radicals generated in a chemical system [Articolo su rivista]
Iannone, Anna; C., Rota; Bergamini, Stefania; Tomasi, Aldo; L. M., Canfield
abstract

b-Carotene is thought to be a chainbreaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of b-carotene and lutein on the radical adducts of the spin-trap PBN (N-t-butyl-a-phenylnitrone) generated by the metal-ion breakdown of different tert-butyl hydroperoxide (tBOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of tBOOH (1 M), were quenched by b-carotene or lutein, in competition with the spintrapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low tBOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by b-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence ofatocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of b-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems.

1998 - Decrease in mitochondrial energy couplings by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence [Articolo su rivista]
Bobyleva, V; Pazienza, Tl; Maseroli, R; Tomasi, Aldo; Salvioli, S; Cossarizza, Andrea; Franceschi, C; Skulachev, Vp
abstract

The effect of the in vivo thyroid status on mitochondrial membrane potential (Delta Psi(m)) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the Delta Psi(m)-specific probe JC-1. It is shown that the Delta Psi(m) level decreases in the order hypothyroid > euthyroid > hyperthyroid, Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6-Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on Delta Psi(m) and respiration rate. Under the same conditions, 6-ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP, The mechanism and possible physiological functions of the thyroid hormone-induced decrease in mitochondrial energy coupling are discussed. (C) 1998 Federation of European Biochemical Societies.

1998 - Le melanocortine come salvavita in gravi condizioni ipossiche caratterizzate da iperproduzione di radicali liberi. [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Bini, Anna; Cainazzo, Maria Michela; Mattera Ricigliano, G.; Tomasi, Aldo
abstract

Le melanocortine come salvavita in gravi condizioni ipossiche caratterizzate da iperproduzione di radicali liberi.

1998 - “The highly reducing sugar 2-deoxy-d-ribose induces apoptosis in human fibroblasts by reduced glutathione depletion and cytoskeletal disruption” [Articolo su rivista]
D., Kletsas; D., Barbieri; D., Stathakos; B., Botti; Bergamini, Stefania; Tomasi, Aldo; D., Monti; W., Malorni; C., Franceschi
abstract

2-deoxy-D-Ribose (dRib), the most reducing sugar, induces apoptosis in normal human fibroblasts, as judged by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation and mitochondrial depolarization. This effect is independent from culture conditions, such as cell density and the presence or absence of serum in the culture milieu, suggesting that dRib-induced apoptosis is cell cycle-independent. dRib was found also to provoke disruption of the actin filament network and detachment from the substratum, while at the same time, interestingly, it increases the expression of several integrins and cell adhesion molecules. Furthermore, dRib was found to reduce the intracellular levels of reduced glutathione (GSH). The apoptotic process was not affected by the macromolecular-synthesis inhibitors cycloheximide and actinomycin D. On the contrary, the antioxidant N-acetyl-L-cysteine (NAG) fully blocks the dRib-induced apoptosis by preventing GSH depletion, while it also inhibits actin-filament-network disruption and mitochondrial depolarization. The above indicate that dRib induces apoptosis in human fibroblasts by a mechanism involving glutathione metabolism and oxidative stress, as well as disturbance of cytoskeletal integrity and cell adhesion.

1997 - Adrenocorticotropin normalizes the blood levels of nitric oxide in hemorrhage-shocked rats [Articolo su rivista]
Guarini, Salvatore; Bini, Anna; Bazzani, Carla; Gm, Ricigliano; Cainazzo, Maria Michela; Tomasi, Aldo; Bertolini, Alfio
abstract

Anesthetized rats were subjected to volume-controlled hemorrhagic shock by stepwise bleeding. Besides cardiovascular and respiratory functions, nitric oxide (NO)-hemoglobin formation in arterial blood was directly evaluated by means of electron spin resonance spectroscopy. During hemorrhagic shock there was a massive increase in NO-hemoglobin, associated with a fall in mean arterial pressure, pulse pressure, respiratory rate and heart rate, and there was a further increase in NO-hemoglobin 15 min after intravenous (i.v.) treatment with saline. All rats died within 30 min. The reversal of the shock condition induced by the i.v. injection of the adrenocorticotropin (ACTH) fragment 1-24 (160 mu g/kg, 5 min after bleeding termination) was associated with a prompt disappearance of NO-hemoglobin. Also S-methylisothiourea (3 mg/kg i.v.), a selective inhibitor of inducible NO synthase, provoked a disappearance of NO-hemoglobin and reversal of the shock condition. The present results provide a direct demonstration that volume-controlled hemorrhagic shock is associated with highly increased blood levels of NO, as indicated by increased NO-hemoglobin, and indicate that ACTH-induced reversal of the shock condition is associated with the normalization of NO blood levels, and a parallel improvement of cardiovascular and respiratory functions. This occurs probably through the inhibition of inducible NO synthase, as suggested by the fact that S-methylisothiourea, a selective inhibitor of this NO synthase isoform, produced the same results.

1997 - Detection of free radicals produced from the reaction of cytochrome P-450 with linoleic acid hydroperoxide [Articolo su rivista]
C., Rota; D. P., Barr; M. V., Martin; F. P., Guengerich; Tomasi, Aldo; R. P., Mason
abstract

The ESR spin-trapping technique was employed to investigate the reaction of rabbit cytochrome P-450 1A2 (P450) with linoleic acid hydroperoxide. This system was compared with chemical systems where FeSO4 or FeCl3 was used in place of P450. The spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) Was employed to detect and identify radical species. The DMPO adducts of hydroxyl, O-2(-.), peroxyl, methyl and acyl radicals were detected in the P450 system. The reaction did not require NADPH-cytochrome P-450 reductase or NADPH. The same DMPO-radical adducts were detected in the FeSO4 system. Only DMPO-(OH)-O-. radical adduct and carbon-centred radical adducts were detected in the FeCl3 system. Peroxyl radical production was completely O-2-dependent. We propose that polyunsaturated fatty acids are initially reduced to form alkoxyl radicals, which then undergo intramolecular rearrangement to form epoxyalkyl radicals. Each epoxyalkyl radical reacts with O-2, forming a peroxyl radical. Subsequent unimolecular decomposition of this peroxyl radical eliminates O-2(-.) radical.

1997 - Inhibition of nitric oxide overproduction, directly detected in the blood by ESR spectrometry, is involved in the mechanism of action of ACTH-(1-24) in hemorrhagic shock reversal. [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Bini, Anna; Tomasi, Aldo; Bertolini, Alfio
abstract

Inhibition of nitric oxide overproduction, directly detected in the blood by ESR spectrometry, is involved in the mechanism of action of ACTH-(1-24) in hemorrhagic shock reversal.

1997 - The rat liver during carbontetrachloride and 1,2-dibromoethane intoxication: a study on biochemical injury and energy state alteration [Articolo su rivista]
Tomasi, A.; Bini, A.; Ghelli, S.; Iannone, Anna; Meletti, E.; Vannini, U. MUSCATELLO AND V.
abstract

The rat liver during carbontetrachloride and 1,2-dibromoethane intoxication: a study on biochemical injury and energy state alteration

1997 - The role of desferrioxamine chelatable iron in rat liver mitochondrial dysfunction in chronic dietary iron overload [Articolo su rivista]
Ceccarelli, D; Kozlov, Av; Gallesi, D; Tomasi, Aldo; Giovannini, F; Masini, A.
abstract

Lipid peroxidation and organelle dysfunction are important factors in hepatic iron toxicity. The form of the intracellular iron responsible for these abnormalities is still unknown. In order to investigate the iron species inducing cell injury, the level of chelatable iron in the liver mitochondria isolated from rats fed a 2.5% carbonyl iron diet for 12 weeks was measured by EPR spectroscopy. The presence of lipid peroxidation products and the energy transducing capability of the mitochondrial inner membrane was evaluated in parallel. The total iron concentration in the liver mitochondria from iron fed rats progressively increased up to 6 weeks, almost reaching a steady-state. By contrast the level of chelatable iron in mitochondrial fraction transiently increased at about 3-6 weeks of treatment. The induction of lipid peroxidation and a large decrease of ATP occurred at the same time. The enhancement of the energy dissipating calcium cycling was in parallel revealed by studying the mitochondria membrane potential. These results gave experimental evidence to the proposal that the chelatable iron level plays a critical role in initiating organelle dysfunction, at least in this experimental model.

1996 - 'Free' iron, as detected by electron paramagnetic resonance spectroscopy, increases unequally in different tissues during dietary iron overload in the rat [Articolo su rivista]
A. V., Kozlov; Bini, Anna; D., Gallesi; F., Giovannini; Iannone, Anna; A., Masini; E., Meletti; Tomasi, Aldo
abstract

'Free' iron concentration, as determined by electron paramagnetic resonance (EPR) spectroscopy, and lipid peroxidation (LPO), as determined by thiobarbituric acid test, were assessed in the lung, heart, liver, spleen, brain and kidney of rats subjected to experimental iron overload, Two tests, Desferal- and NO-available iron, were used to measure 'free' iron and gave comparable results, The most pronounced accumulation of 'free' iron was observed in liver, kidney and spleen, Differences between control and iron loaded animals increased during the initial 90 days of treatment, Between 90 and 180 days 'free' iron concentration reached a steady state level, or even decreased, as in the case of liver, Lipid peroxidation level, measured in the organs of both treated and matched controls, did not give any significant difference during the initial 90 days of treatment, A significant augmentation was observed in liver, kidney, spleen and heart at 180 days, The results of the present research show that, under conditions of moderate siderosis, the occurrence of LPO is partially related to the level of 'free' iron.

1996 - Age-related differences in the metabolism of sulphite to sulphate and in the identification of sulphur trioxide radical in human polymorphonuclear leukocytes [Articolo su rivista]
Constantin, D; Bini, A; Meletti, E; Moldeus, P; Monti, D; Tomasi, Aldo
abstract

Sulphite oxidation and sulphur trioxide radical formation were studied in polymorphonuclear leukocytes (PMNs) isolated from healthy young, old and centenarian donors and from patients with Down's syndrome. The sulphur radical formation measured by electron spin resonance spectroscopy-spin trapping (EPR-ST) was correlated with the activity of sulphite oxidase and with the rate of sulphite oxidation to sulphate by PMNs. Sulphite metabolism was studied both in resting, and phorbol myristate acetate (PMA) stimulated freshly isolated cells. The rate of sulphur trioxide radical formation was demonstrated by use of the spin trapping agent 5,5-dimethyl-1-pyroline-1-oxide (DMPO) with subsequent formation of an adduct. The intensity of adduct formation was most intense in cells with low sulphite oxidase activity, while a mixture of the adduct and of DMPO hydroxyl radical was mainly observed in cells with high sulphite oxidase activity. Furthermore, experiments carried out on purified sulphite oxidase showed that in the presence of sulphite the enzyme could also give rise to a DMPO-OH adduct. Sulphite oxidase activity in cells isolated from healthy young and old donors was positive correlated with both rates of sulphur trioxide radical formation and sulphite oxidation to sulphate, respectively. However, sulphite oxidase activity in cells isolated from centenarians and patients with Down's syndrome seems to loose partly its rate of oxidising sulphite to sulphate. The intensity of the sulphur centred radical adduct increased in the two latter groups of population and the radical observed was predominantly sulphur trioxide radical.

1996 - Dietary fatty acids composition modulates oxidative stress and cellular injury in the liver of CBrCl3 intoxicated rats [Articolo su rivista]
F., Virgili; Battistini, Nino Carlo; Bini, Anna; V., Vannini; Tomasi, Aldo
abstract

Oxidative stress is a multifactorial event that has been proposed to be involved in the aethyology of a large number of human pathologies. The fatty acid composition of dietary lipid has been reported as one of major factors capable of modulating oxidative stress. In this paper we assess the effect of different types of dietary lipids on microsome fatty acids composition and on the oxidative stress induced by the administration of CBrCl3. Three groups of rats were fed different experimental diets providing 20% of energy from lipid derived by olive, coconut, and sunflower oils. Membrane fatty acid composition and antioxidant vitamins was determined at the end of six weeks diet. The dietary induced changes of membrane fatty acids composition were associated with different susceptibility to oxidative stress and cell damage, as assessed by conjugated dienes and cytosolic enzyme release respectively following CBrCl3 intoxication. Feeding the CO diet leads to a low susceptibility to lipid peroxidation, but is associated with cell injury significantly higher than that observed in the other dietary treatments. No association was demonstrated between the extent of membrane lipid peroxidation and liver cell damage.

1996 - Electron paramagnetic resonance characterization of rat neuronal nitric oxide production ex vivo [Capitolo/Saggio]
Av, Kozlov; Bini, Anna; Iannone, Anna; Zini, Isabella; Tomasi, Aldo
abstract

n.d.

1996 - Generation of N-tert-butyl-alpha-phenylnitrone radical adducts in iron breakdown of tert-butyl-hydroperoxide [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo; Canfield, Lm
abstract

ESR spectroscopy coupled to the spin trapping technique was used to evaluate the generation of radical species arising from the ferrous ion induced decomposition of tert-butyl hydroperoxide ('BuOOH) in methylene chloride. We report here that N-tert-butyl-alpha-phenylnitrone (PEN) can trap peroxyl radicals generated in the ferrous ion induced breakdown of high concentration of 'BuOOH (1M) at room temperature, the radical adduct being stable under the light. The peroxyl radical formation was demonstrated by direct ESR measurements at 77K. In contrast, alkoxyl and methyl radicals were trapped only in the presence of low hydroperoxide concentration (1mM). In order to measure the hyperfine splitting constants (hfsc) of the PEN-methyl adduct spectra were obtained in the presence of diphenylamine (DPA) or 2,6-di-tert-butyl-4-methylphenol (BHT), which quenched the alkoxyl radical. For this latter radical, the hfsc were calculated by computer simulation. A mechanism for a direct interaction between DPA and the alkoxyl radical is presented. DPA quenched the peroxyl radical in the reaction of high hydroperoxide concentrations, with the concomitant generation of a DPA nitrogen-based radical.

1996 - High glucose-induced membrane lipid peroxidation on intact erythrocytes and on isolated erythrocyte membrane (ghosts) [Articolo su rivista]
Virgili, F; Battistini, Nino Carlo; Canali, R; Vannini, V; Tomasi, Aldo
abstract

Glucose autoxidation has been proposed to play a role in diabetes complications via free radicals damage to cells, but the mechanisms that should link high glucose to oxidative stress is stress is still partially unknown. In this study we report that high glucose induces lipid peroxidation, membrane tocopherol consumption, and lysis in intact erythrocytes (RBC), whereas antioxidant enzyme activity was not affected. On the contrary, no lipid peroxidation or free thiols oxidation was detectable in isolated erythrocyte membranes (ghosts) incubated either with glucose or methyl-glyoxal, a carbonyl molecule, non-enzymatic byproduct of high glucose metabolism. Our study indicates that the presence of transition metals is the necessary condition to induce glucose-driven lipid peroxiation and thiols oxidation. This study also suggests that the incubation of RBC with glucose concentration similar to non-controlled hyperglycaemia may led to the release of iron in redox active form.

1996 - Influence of ACTH-(1-24) on free radical levels in the blood of haemorrhage-shocked rats: Direct ex vivo detection by electron spin resonance spectrometry [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Gm, Ricigliano; Bini, Anna; Tomasi, Aldo; Bertolini, Alfio
abstract

1 The influence of ACTH-(1-24) on the blood levels of highly reactive free radicals in haemorrhagic shock was studied in rats. 2 Volume-controlled haemorrhagic shock was produced in adult rats under general anaesthesia (urethane, 1.25 g kg(-1) intraperitoneally) by stepwise bleeding until mean arterial pressure stabilized at 20-23 mmHg. Rats were intravenously (i.v.) treated with either ACTH-(1-24) (160 mu g kg(-1) in a volume of 1 ml kg(-1)) or equivolume saline. Free radicals were measured in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent (alpha-phenyl-N-tert-butylnitrone). 3 Blood levels of free radicals were 6490+/-273 [arbitrary units (a.u.) ml(-1) whole blood, before starting bleeding, and 30762+/-2650 after bleeding termination (means+/-s.e.mean of the values obtained in all experimental groups). All rats treated with saline died within 30 min, their blood levels of free radicals being 35450+/-5450 a.u. ml(-1) blood, 15 min after treatment. Treatment with ACTH-(1-24) produced a rapid and sustained restoration of arterial pressure, pulse pressure, heart rate and respiratory function, with 100% survival at the end of the observation period (2 h); this was associated with an impressive reduction in the blood levels of free radicals, that were 12807+/-2995, 10462+/-2850, 12294+/-4120, and 10360+/-2080 a.u. ml(-1) blood, 15, 30, 60 and 120 min after ACTH-(1-24) administration, respectively. 4 These results provide a direct demonstration that (i) in haemorrhagic shock there is a rapid and massive production of highly reactive free radicals, and that (ii) the sustained restoration of cardiovascular and respiratory functions induced by the i.v. injection of ACTH-(1-24) is associated with a substantial reduction of free radical blood levels. It is suggested that ACTH-(1-24) prevents the burst of free radical generation during blood mobilisation and subsequent tissue reperfusion, and this may be an important component of its mechanism of action in effectively preventing death for haemorrhagic shock.

1996 - No direct evidence of increased lipid peroxidation in hemodialysis patients [Articolo su rivista]
S., Banni; L., Lucchi; A., Baraldi; B., Botti; Cappelli, Gianni; F., Corongiu; Ma, Dessi; Tomasi, Aldo; E., Lusvarghi
abstract

Lipid peroxidation, as measured by the thiobarbituric acid test, has been reported to have increased in hemodialysis (HD) patients, even though the test has low specificity in vivo. Conjugated diene fatty acid (CDFA) hydroperoxides are formed during lipid peroxidation, but not all conjugated dienes (CD) detected in humans originate from lipid peroxidation: octadeca-9,11-dienoic acid, a nonhydroperoxide CD derivative of linoleic acid (CDLA), has a dietary origin. We evaluated CDFA hydroperoxides, CDLA and linoleic acid, using high-performance liquid chromatography, in lipids extracted from plasma, adipose tissue and RBC membranes obtained from 25 patients treated with HD, 16 patients treated with hemodiafiltration (HDF) and 29 controls. No differences in the levels of CDFA hydroperoxides and linoleic acid were seen in any of the groups. Concentrations of CDLA were found to be significantly high in the adipose tissue and low in the RBC membranes of HD patients. HDF-treated patients showed the same results as HD patients. No direct evidence of increased lipid peroxidation was found in HD patients. This does not exclude the possibility that lipid peroxidation is increased and escapes direct detection due to the body's homeostatic control eliminating the increased production of hydroperoxides. Both HD- and HDF-treated patients showed a significant change in CDLA concentrations, either in the adipose tissue, or in the RBC membranes. These dietary CD may be mistaken for markers of lipid peroxidation by conventional methodologies.

1996 - Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol [Articolo su rivista]
Albano, E; Clot, P; Morimoto, M; Tomasi, Aldo; Ingelmansundberg, M; French, Sw
abstract

We have previously shown that the treatment with diallyl sulfide (DAS) and phenylethyl isothiocyanate (PIG) of rats receiving ethanol in the alcohol tube-feeding model effectively suppressed the induction of cytochrome P4502E1 (CYP2E1) by ethanol, Here we report that rat treatment with DAS and PIC significantly decreased the trapping of hydroxyethyl free radicals in Liver microsomes incubated in vitro with ethanol. Furthermore, these inhibitors also greatly reduced the production of hydroxyethyl radical derived epitopes detectable in vivo in the liver of ethanol-fed rats. The action of DAS and PIC on the formation of hydroxyethyl radicals paralleled their inhibitory effect on lipid peroxidation as monitored using, respectively, liver malonildialdehyde (MDA) and plasma lipid hydroperoxide levels as well as by the titers of antibodies versus MDA adducts to proteins, Thus, these results indicated a Link between the induction of CYP2E1 by ethanol, the formation of hydroxyethyl radicals and the stimulation of lipid peroxidation, The pathological scores in the Livers of rats fed with ethanol plus or minus DAS and PLC also correlated with levels of hydroxyethyl radical-derived epitopes, Rats fed intragastrically with ethanol developed antibodies reacting with hydroxyethyl radicals-modified proteins and the formation of these antibodies was greatly reduced by DAS and PIG. Taken together these results suggest that CYP2E1 plays an important role in the generation of hydroxyethyl radicals during chronic alcohol feeding and that ethanol-derived free radicals might play a role in the onset of Liver injury in this model of alcohol administration.

1996 - The ACTH-induced reversal of haemorrhagic shock is associated with an impressive reduction of free radical and nitric oxide levels in blood [Abstract in Rivista]
Bazzani, Carla; Guarini, Salvatore; Bini, Anna; Mattera Ricigliano, G.; Meletti, Eros; Tomasi, Aldo; Bertolini, Alfio
abstract

The ACTH-induced reversal of haemorrhagic shock is associated with an impressive reduction of free radical and nitric oxide levels in blood

1996 - The resuscitating effect of ACTH-(1-24) after prolonged respiratory arrest is associated with normalization of nitric oxide and free radical blood levels. [Abstract in Rivista]
Guarini, Salvatore; Bini, Anna; Bazzani, Carla; Cainazzo, M. M.; Mattera Ricigliano, G.; Tomasi, Aldo; Bertolini, Alfio
abstract

The resuscitating effect of ACTH-(1-24) after prolonged respiratory arrest is associated with normalization of nitric oxide and free radical blood levels.

1995 - Actvation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P4502E1 (CYP2E1) [Articolo su rivista]
Albano, E; Comoglio, A; Clot, P; Iannone, Anna; Tomasi, Aldo; INGELMAN SUNDBERG, I.
abstract

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, beta-naphthoflavone (beta-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.

1995 - Ex-vivo demonstration of nitric oxide in the rat brain: Effects of intrastriatal endothelin-1 injection [Articolo su rivista]
A. V., Kozlov; Biagini, Giuseppe; Tomasi, Aldo; Zini, Isabella
abstract

Nitric oxide (NO.) is a novel transmitter with multiple functions in endothelium and neuronal tissue. In particular, it has been implicated in the pathogenesis of neurodegenerative diseases. The aim of the present study was to demonstrate the ex vivo detection Df NO. in basal conditions and after ET-1 intrastriatal injection by means of electron paramagnetic resonance (EPR) spectroscopy using locally injected hemoglobin (Hb) as a NO. trapping agent. The extent of neostriatal damage after Hb and ET-1 injections was assessed by means of immunocytochemistry with a monoclonal antibody against dopamine and cAMP-phosphoprotein M(r) 32 (DARPP-32), which is considered a marker of striatal intrinsic neurons. In the absence of local Hb injection, no signal related to endogenous NO. was detected in the neostriatum, suggesting that endogenous NO. trapping agents are not sufficiently concentrated to allow NO. detection with the present technique. Instead, 1 h after Hb injection, a clear nitrosyl-Hb signal can be detected in neostriatal homogenates. ET-1, a powerful vasoconstrictor agent, was used to cause neuronal loss in the neostriatum. No change in nitrosyl-Hb signal was observed in neostriata 1 h after ET-1 injection, whereas an almost 3-fold increase in the signal intensity was present 24 h after ET-1 injection. The analysis of neostriatal damage showed that Hb injection did not cause either significant damage of striatal tissue or potentiation of ET-1-induced lesions. In conclusion, the present technique allows ex vivo detection of NO. in the brain. The delayed increase in NO. observed after ET-1 injection indicates that this molecule may participate in the development of slowly progressive neuronal damage occurring at late post-ischemic times.

1995 - INDUCTION OF FERRITIN SYNTHESIS BY OXIDATIVE STRESS - TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION BY EXPANSION OF THE FREE IRON POOL [Articolo su rivista]
Cairo, G; Tacchini, L; Pogliaghi, G; Anzon, E; Tomasi, Aldo; Bernellizazzera, A.
abstract

Ferritin, by regulating the ''free'' intracellular iron pool, controls iron-catalyzed generation of reactive oxygen species, but its role in oxidative damage is still unclear. We show that ferritin synthesis is significantly stimulated in the liver of rats subjected to oxidative stress by treatment with phorone, a glutathione-depleting drug, RNA-bandshift assays document reduced activity of iron regulatory factor, in particular of IRF(B), the cytoplasmic protein that post-transcriptionally controls ferritin mRNA translation, Furthermore, Northern blot analysis shows increased accumulation of H and L subunit mRNAs, and nuclear run-on experiments provide evidence of transcriptional activation, Direct measurements of intracellular free iron levels by EPR indicate that the increased ferritin synthesis can be mediated by an expansion of the free iron pool, An early drop of ferritin content after phorone treatment indicates that part of the iron that fuels the free pool might derive from ferritin degradation, Present data seem to suggest that, under conditions of oxidative stress, liver ferritin can represent either a pro- or an anti-oxidant in a time-dependent manner, In fact, its early degradation contributes to expand the intracellular free iron pool that, later on, activates multiple molecular mechanisms to reconstitute ferritin content, thus Limiting the prooxidant challenge of iron.

1995 - SCAVENGING EFFECT OF SILIPIDE, A NEW SILYBIN-PHOSPHOLIPID COMPLEX, ON ETHANOL-DERIVED FREE-RADICALS [Articolo su rivista]
Comoglio, A; Tomasi, Aldo; Malandrino, S; Poli, G; Albano, E.
abstract

Ethanol metabolism by cytochrome P4502E1 (CYP2E1) produces free radical intermediates, identified as hydroxyethyl radicals. We have observed that in vitro addition or in vivo pretreatment of rats with Silipide, a new 1:1 complex of silybin with phosphatidyl-choline, is able to decrease the spin trapping of hydroxyethyl radicals in microsomes from chronic alcohol-fed rats. This effect is not due to an interference with the metabolism of ethanol by CYP2E1, but is rather related to the capacity of the silybin molecule to scavenge hydroxyethyl radicals. However, such an effect is lost when pure silybin in amounts comparable to those present in Silipide is administered instead, due to the low bioavailability of uncomplexed flavonoid. Further experiments in vivo have shown that Silipide administration also decreases hydroxyethyl radical signals detectable in the bile of rats acutely treated with ethanol. The ability of Silipide to scavenge ethanol-derived radicals along with its antioxidant activity suggests that this drug might be potentially useful in counteracting free radical-mediated injuries involved in the development of liver damage caused by alcohol abuse.

1994 - ALTERNATIVE PATHWAYS OF SULFITE OXIDATION IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES [Articolo su rivista]
Constantin, D; Mehrotra, K; Jernstrom, B; Tomasi, Aldo; Moldeus, P.
abstract

Sodium sulfite is metabolized by human polymorphonuclear leukocytes by two alternative pathways, one enzymatic route dependent on sulfite oxidase and one non-enzymatic which involves intermediate formation of sulfur trioxide anion radicals. Initiation of the oxidative burst by phorbol myristate acetate significantly stimulates sulfate formation through the second pathway. The activity of sulfite oxidase in polymorphonuclear leukocytes varies greatly among individuals, a variation consistent with the suggested polymorphic distribution of sulfite oxidase in the human population.

1994 - FREE-RADICAL ACTIVATION OF ACETALDEHYDE AND ITS ROLE IN PROTEIN ALKYLATION [Articolo su rivista]
Albano, E; Clot, P; Comoglio, A; Dianzani, Mu; Tomasi, Aldo
abstract

The formation of carbon centered free radicals, identified as methylcarbonyl species, was observed using ESR spectroscopy and the spin trapping agent 4-pyridyl-1-oxide-N-t-butyl nitrone (4-POBN) during the oxidation of acetaldehyde by xanthine oxidase. The reaction was dependent upon the presence of OH* radicals and was inhibited by the addition of superoxide dismutase, catalase or OH* radical scavengers. The generation of methylcarbonyl radicals was associated with a doubling of stable acetaldehyde adducts with serum albumin, and 4-POBN or superoxide dismutase and catalase, completely blocked this effect. Thus, methylcarbonyl radicals contributed to acetaldehyde-mediated protein alkylation which is involved in causing toxic as well as immunological reactions ascribed to acetaldehyde.

1994 - Hydroperoxide metabolism in cultured normal human Keratinocytes a spin trapping study [Capitolo/Saggio]
Giannetti, Alberto; Marconi, Alessandra; G., Zambruno; Iannone, Anna; Tomasi, Aldo
abstract

.

1994 - SPIN-TRAPPING OF ALCOHOL-DERIVED RADICALS IN MICROSOMES AND RECONSTITUTED SYSTEMS BY ELECTRON-SPIN-RESONANCE [Capitolo/Saggio]
Albano, E; Tomasi, Aldo; Ingelmansundberg, M.
abstract

*Alcohols metabolism; *Cytochrome P 450 Enzyme System metabolism; *Electron Spin Resonance Spectroscopy methods; *Microsomes, Liver metabolism; *Spin LabelsAlcohols analysis; Cytochrome P 450 Enzyme System antagonists and inhibitors; Free Radicals analysis; Indicators and Reagents; NADP metabolism; Nitrogen Oxides; Oxidation Reduction; Rats ; Rats, Sprague Dawley; Rats, Wistaranalysis; metabolism; antagonists and inhibitors; methodsCAS: 0; 0; 0; 0; 0; 0; 53 59 8; 9035 51 2

1993 - CONJUGATED DIENE FATTY-ACIDS IN PATIENTS WITH CHRONIC-RENAL-FAILURE - EVIDENCE OF INCREASED LIPID-PEROXIDATION [Articolo su rivista]
Lucchi, L; Banni, S; Botti, B; Cappelli, Gianni; Medici, G; Melis, Mp; Tomasi, Aldo; Vannini, V; Lusvarghi, E.
abstract

Conjugated diene fatty acids (CDFA) were evaluated by second derivative spectrophotometry in the plasma and adipose tissue of 42 chronic renal failure (CFR) patients in conservative treatment, 40 patients treated by hemodialysis (HD) with cuprophane, cellulose acetate or hemophan, 29 treated by hemodiafiltration (HDF) with polysulfone, polyacrylonitrile or polyamide, and 28 healthy controls. Plasma CDFA were also evaluated at the beginning, at 30 min and at the end of the dialytic session. CDFA were unchanged in CRF patients with creatinine clearance (Ccr) > 10 ml/min respect to the controls, CRF patients with Ccr < 10 ml/min showed a higher level of CDFA both in plasma and adipose tissue (p < 0.02). HD patients showed values similar to those of the control group. The lowest level of CDFA was found in HDF patients (p < 0.01 for plasma, p < 0.05 for adipose tissue versus both control and any other group). A significant relationship between plasma and adipose tissue CDFA was found in all groups? In the group of CRF patients with Ccr < 10 ml/min, females exhibited a higher level of CDFA both in plasma and adipose tissue. No significant change was found during dialytic session, independently from the membrane used. CDFA are not only primary products of lipid peroxidation, but also have a dietary origin, primarily from dairy products. Taking into account the reduced dietary intake, the increase in end-stage CRF may be due to an enhanced oxidative stress and/or to abnormalities in CDFA metabolism. Uremic patients, particularly in the predialytic stage, should be considered at risk for increased oxidative stress. HDF treatment better corrects the abnormality compared to conventional HD.

1993 - Free-Radical Production During Metabolism Of Organic Hydroperoxides By Normal Human Keratinocytes [Articolo su rivista]
Iannone, Anna; Marconi, Alessandra; G., Zambruno; Giannetti, Alberto; V., Vannini; Tomasi, Aldo
abstract

Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.

1993 - Nitroxides as metabolic and EPR imaging probes in biological model systems [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo; Quaresima, V; Ferrari, M.
abstract

Nitroxides are unusually stable free radicals and sensitive ''reporters'' of the chemico-physical environment which surround them. The term ''reporter group'' was used to indicate this property, later the term spin label or spin probe was preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications. Nitroxides have been used to study molecular mobility of proteins and lipids in membranes, enzyme active sites, DNA synthesis, and measure of electrical potential, pH, temperature as well as oxygen gradients across membranes; more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging (EPRI). Aim of this work is to review and comment on the use of various nitroxides as metabolic probes, and in EPRI in studies of biomedical interest.

1993 - Possible role of free radical intermediates in hepatotoxicity of hydrazines derivatives [Articolo su rivista]
Albano, E; Goriagatti, L; Clot, P; Iannone, Anna; Tomasi, Aldo
abstract

Hydrazine derivatives constitute a wide group of compounds and have found application in industry, agriculture, and (as therapeutical agents) medicine. In spite of their widely spread use, several hydrazine derivatives are known to exert hepatotoxic effects and are carcinogenic. Free radical species are produced during the hepatic biotransformation of alkylhydrazines by both rat and humans liver microsomes. Cytochrome P-450 dependent monoxygenase system is responsible for the production of these reactive species and specific cytochrome P-450 isoenzymes appear to catalyze the formation of free radical intermediates. Free radicals generated during the metabolism of alkylhydrazines are capable of inducing oxidative stress in isolated hepatocytes and might contribute to the development of cell injury.

1993 - t-Butyl-hydroperoxide bioactivation to methyl radical in rat liver mitochondria and submitochondrial particles [Articolo su rivista]
Iannone, Anna; Bini, A; JIN Y., G; Tomasi, Aldo; Vannini, V.
abstract

Electron paramagnetic resonance spectroscopy (EPR) coupled to the spin trapping technique was used to detect carbon-centered radicals in rat liver mitochondria and submitochondrial particles exposed to t-butyl-hydroperoxide (TBH), using the spin trapping agent 3,5-dibromo-4-nitroso-benzenesulfonic acid (DBNBS). The signal recorded was unambiguously assigned to the methyl radical adduct. DBNBS was added to isolated rat liver mitochondria energized with succinate, and the methyl radical adduct was observed. The addition of NADH, NADPH, inhibitors of the respiratory chain, and of monoaminoxidase (MAO) inhibitors did not cause any relevant modification in the yield of radical adduct formation. Boiling and the addition of a non-ionic detergent inhibited the formation of the radical adduct, while experiments carried out under hypoxic conditions generated a significant increase in methyl radical formation. Further experiments were carried out on sub-mitochondrial particles (SMP) giving rise to, basically, the same results. From the above results, we are proposing that haem prosthetic groups are the likely source of TBH bioactivation in mitochondria.

1992 - DETECTION AND QUANTITATION IN RAT-TISSUES OF THE SUPERPARAMAGNETIC MAGNETIC-RESONANCE CONTRAST AGENT DEXTRAN MAGNETITE AS DEMONSTRATED BY ELECTRON-SPIN-RESONANCE SPECTROSCOPY [Articolo su rivista]
Iannone, Anna; Federico, Massimo; Tomasi, Aldo; Magin, Rl; Casasco, A; Calligaro, A; Vannini, V.
abstract

RATIONALE AND OBJECTIVES. The compound studied in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. METHODS. DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. RESULTS. DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. CONCLUSIONS. ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.

1992 - DETECTION OF FREE-RADICALS DURING BRAIN ISCHEMIA AND REPERFUSION BY SPIN TRAPPING AND MICRODIALYSIS [Articolo su rivista]
Zini, Isabella; Tomasi, Aldo; Grimaldi, R; Vannini, V; Agnati, Luigi Francesco
abstract

Extracellular free radicals were detected in rat striatal perfusate samples by intracerebral microdialysis coupled to the spin trapping technique. Five Sprague-Dawley rats were subjected to 30 min of global ischemia followed by reperfusion; throughout the experimental period the intrastriatal dialysing probe was perfused with Ringer's solution containing the spin trap agent pyridyl-N- oxide-t-butylnitrone (100 mM) together with the iron chelating agent diethylenetriaminepentaacetic acid (100-mu-M). A radical adduct occurred during ischemia and early reperfusion, but not in basal conditions; the spin adduct was characterized as a carbon centered radical, consistent with the presence of an oxidative attack on membrane lipids. The direct evidence of the formation of free radicals supports the hypothesis that free radicals play a role in the pathogenesis of the histological damage during brain ischemia.

1992 - In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin trapping study [Articolo su rivista]
GORIA GATTI, L; Iannone, Anna; Tomasi, Aldo; Poli, G; Albano, E.
abstract

Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.

1991 - Blood clearance of dextran magnetite particles by a non invasive in vivo ESR method [Articolo su rivista]
Iannone, Anna; Magin, R. L.; Walczac, T.; Federico, Massimo; Swartz, H. M.; Tomasi, Aldo; Vannini, V.
abstract

Dextran magnetite (DM) is a potential MR contrast agent with superparamagnetic properties. Its fast clearance from the blood and selective uptake by tissue macrophages provide advantages for imaging tumors in the liver and spleen. DM consists of a suspension of solid particles with a wide distribution of sizes. In this study we have used ESR spectroscopy to determine the blood clearance of DM injected iv in mice. The spectra are obtained on living animals by inserting the tail of a mice into the waveguide cavity of the ESR spectrometer and recording the ESR spectrum continuously. This procedure allows the direct measurement of the plasma clearance of DM from individual animals, without blood sampling. We applied this method to study the clearance of suspensions of DM particles with different average sizes.

1991 - ENZYMATIC RECYCLING OF OXIDIZED ASCORBATE IN PIG-HEART - ONE-ELECTRON VS 2-ELECTRON PATHWAY [Articolo su rivista]
Coassin, M; Tomasi, Aldo; Vannini, V; Ursini, F.
abstract

Enzymatic systems able to reduce either dehydroascorbate or ascorbyl radical back to ascorbate by "recycling" vitamin C may contribute to lowering the nutritional requirement of it and to increase tissue antioxidant capacity. The activities of two enzymatic activities, GSH-dehydroascorbate reductase (two-electron reduction pathway) and NADH-semidehydroascorbate reductase (one-electron reduction pathway) in pig tissues, have been investigated. The activity of glutathione-dependent reduction of dehydroascorbate, although measurable, appeared negligible taking into consideration the low physiological substrate concentration. On the other hand, the one-electron reduction of ascorbyl radical resulted fast enough to slow down the consumption of the antioxidant vitamin.

1991 - NITROXIDE RADICALS, THEIR USE AS METABOLIC PROBES IN BIOLOGICAL MODEL SYSTEMS - AN OVERVIEW [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo
abstract

The concept of using a probe, sensitive to the environment, termed >>reporter group<<, arose in the early sixties; readily, nitroxides became the natural candidate for a >>reporter group<<. The term >>reporter group<< was later dismissed and the term spin label or spin probe preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications including molecular mobility of proteins and lipids in membranes, study of the enzyme active site and DNA synthesis, measurement of electrical potential, measurement of pH, temperature and oxygen gradients across membranes and, more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging. The overview, by no means comprehensive of the complete literature, aims to give an insight view of nitroxide applications as metabolic probes in the field of biomedical studies reporting methodologies and trying to give a critical view of the procedures and methodologies used.

1991 - ROLE OF ETHANOL-INDUCIBLE CYTOCHROME-P450 (P450IIE1) IN CATALYZING THE FREE-RADICAL ACTIVATION OF ALIPHATIC-ALCOHOLS [Articolo su rivista]
Albano, E; Tomasi, Aldo; Persson, Jo; Terelius, Y; Goriagatti, L; Ingelmansundberg, M; Dianzani, Mu
abstract

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxpropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.

1990 - Free radical detection during xenobiotics metabolism by human keratinocytes [Capitolo/Saggio]
Giannetti, Alberto; Marconi, Alessandra; M. L., Santantonio; G., Zambruno; Iannone, Anna; Tomasi, Aldo
abstract

.

1990 - Lipid composition and fluidity of liver mitochondria, microsomes and plasma membrane of rats with chronic dietary iron overload. [Articolo su rivista]
Pietrangelo, Antonello; Grandi, R; Tripodi, A; Tomasi, Aldo; Ceccarelli, D; Ventura, E; Masini, A.
abstract

The effect of chronic dietary iron overload on the lipid composition and physical state of rat liver mitochondria, microsomes and plasma membranes was investigated. After 9 weeks of iron treatment, a significant decrease of polyunsaturated and a parallel increase of saturated fatty acids was observed in mitochondrial and plasma membrane phospholipids. By contrast, no appreciable modification of the fatty acid composition of microsomal membranes was detected. The cholesterol/phospholipid molar ratio as well as the lipid/protein ratio, did not reveal any significant difference in any of the fractions studies. Finally, no change in the molecular order of the various membranes, as assessed by electron spin resonance spectrometry, was observed following iron intoxication. These data indicate that, although in vivo chronic hepatic iron overload induces a modification of fatty acid profile in cellular structures consistent with the in vivo occurrence of lipid peroxidation, these changes do not bring about appreciable modifications of other physico-chemical parameters relevant to membrane integrity and cell viability.

1990 - METABOLISM OF NITROXIDE SPIN LABELS IN SUBCELLULAR FRACTION OF RAT-LIVER .1. REDUCTION BY MICROSOMES [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo; Vannini, V; Swartz, Hm
abstract

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.

1990 - METABOLISM OF NITROXIDE SPIN LABELS IN SUBCELLULAR-FRACTIONS OF RAT-LIVER .2. REDUCTION IN THE CYTOSOL [Articolo su rivista]
Iannone, Anna; Tomasi, Aldo; Vannini, V; Swartz, Hm
abstract

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.

1990 - Oxidative stress, poly(ADP)ribosylation and aging: In vitro studies on lymphocytes from normal and Down's syndrome subjects of different age and from patients with Alzheimer's dementia [Relazione in Atti di Convegno]
Franceschi, C.; Monti, D.; Cossarizza, A.; Tomasi, A.; Sola, P.; Zannotti, M.
abstract

1990 - STUDIES ON THE ANTIOXIDANT AND FREE-RADICAL SCAVENGING PROPERTIES OF IDB-1016 A NEW FLAVANOLIGNAN COMPLEX [Articolo su rivista]
Comoglio, A; Leonarduzzi, G; Carini, R; Busolin, D; Basaga, H; Albano, E; Tomasi, Aldo; Poli, Giorgio; Morazzoni, P; Magistretti, Mj
abstract

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has been evaluated. One hour after the intragastric administration to rats of IdB 1016 (1.5 g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5 micrograms/mg protein corresponding to a final concentration in the microsomal suspension used of about 10 microM. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CCl4 and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine. These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.

1989 - Ascorbyl radical is detected in rat isolated hepatocytes suspensions undergoing oxidative stress: an early index of oxidative damage in cells [Relazione in Atti di Convegno]
Tomasi, Aldo; E., Albano; A., Bini; Iannone, Anna; V., Vannini
abstract

In this article we have studied the formation of free radical intermediates induced by paraquat, whose toxicity is well known, and by the anti-tumour agent adriamycin in isolated rat hepatocytes comparing the results to those obtained using an iron chelate, such as ADP-iron, a strong inducer of lipid peroxidation. Both paraquat and adriamycin give rise to toxic reactive species of oxygen, our goal being that of inducing an oxidative stress in the model system used.

1989 - Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes [Articolo su rivista]
E., Albano; Tomasi, Aldo; L., Goria Gatti; Iannone, Anna
abstract

Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.

1989 - Free radicals and lipid peroxidation in liver of rats kept on a diet devoid of choline [Articolo su rivista]
S., Banni; F. P., Corongiu; M. A., Dessì; Iannone, Anna; B., Lombardi; Tomasi, Aldo; V., Vannini
abstract

Rodents kept on a choline devoid (CD) diet up to 14 months develop hepatic lesions progressing through two broad stages. The first is characterized by severe steatosis and increase in cell turnover, the second by a gradual clearance of the deposited fat and fibrosis. Hepatocellular carcinomas eventually arise in rats fed for over 12 months, even though the animals aer not exposed to chemical carcinogens. It has been suggested that the diet may trigger generated thereby may be responsible for initiation of liver cancer and promotion. The radicals would lead to DNA damage, and the altered DNA in a proliferating liver would result in initiation of the carcinogenic process. In this communication we present evidence that the diet used in the above studies contained stable fatty acid isomers with conjugated dienes, which are absorbed and deposited in rat liver. This finding cast doubts on whether a CD diet does indeed cause a peroxidation of cellular membrane lipids. Electron spin resonance (ESR) spectroscopy was also used to investigate whether any abnormal pattern of free radicals exists in the liver of rats fed a CD diet. No significant differences were noted in ESR spectra of either transition metal-centered signals, or organic free radicals.

1989 - Lipid composition and fluidity of liver plasma membranes from rats with chronic dietary iron overload. [Articolo su rivista]
Pietrangelo, Antonello; Tripodi, A; Carulli, N; Tomasi, Aldo; Ceccarelli, D; Ventura, Ezio; Masini, A.
abstract

Liver plasma membranes isolated from rats with chronic dietary iron overload showed a large modification of their phospholipid fatty acid composition. Specifically, a significant decrease in polyunsaturated fatty acids and a parallel increase in saturated fatty acids was observed. This pattern was consistent with the in vivo occurrence of lipoperoxidative reactions in the liver plasma membranes. However, neither change in the cholesterol/phospholipid molar ratio nor in the lipid/protein ratio was detected. Direct measurement of the plasma membrane fluidity state by electron spin resonance spectrometry did not reveal any difference between control and iron-treated rats. These findings indicate that chronic dietary iron overload can induce lipid peroxidation of rat liver plasma membranes, but this event does not bring about modification in the physical state of the membrane.

1989 - Metabolism in rat liver microsomes of the nitroxide spin probe Tempol [Articolo su rivista]
Iannone, Anna; A., Bini; H. M., Swartz; Tomasi, Aldo; V., Vannini
abstract

Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.

1989 - Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides [Articolo su rivista]
Iannone, Anna; H., Hu; Tomasi, Aldo; V., Vannini; H. M., Swartz
abstract

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides

1987 - Detection of Free Radical Intermediates in the Oxidative Metabolism of Carcinogenic Hydrazine Derivatives [Articolo su rivista]
Tomasi, Aldo; E., Albano; B., Botti; V., Vannini
abstract

Hydrazine derivatives are widely used in agriculture, in industry, as rocket propellants, and in medicine. Hydrazines also occur naturally in tobacco and mushrooms. Many hydrazines tested in animal studies appear to be carcinogenic and induce tumors in various target tissues in mice, hamsters, and rats. The use of hydrazine derivatives in humans is often complicated by adverse side-effects such as liver injury and rheumatoid arthritis. A number of studies have demonstrated that hydrazine derivatives are activated to reactive intermediates, such as free radicals, through a variety of cellular oxidative metabolic pathways. The aim of this work is to demonstrate the occurrence of free radical intermediates during the metabolic activation of various hydrazine derivatives and to characterize the enzymatic system(s) responsible for the activation to free radical species. The hydrazines studied are acetylhydrazine, isoniazid, isopropylhydrazine, iproniazid, methylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine. The model systems chosen are those of rat liver microsomes and isolated hepatocytes. Free radical intermediates have been demonstrated by the electron spin resonance spectroscopy coupled to spin trapping technique. The activation mechanism has been characterized using inhibitors of the mixed function oxidase system and of the FAD-dependent oxygenase system. Glutathione was able to scavenge, with high efficiency, the free radicals produced.

1987 - Free radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation [Articolo su rivista]
TOMASI, Aldo; E., Albano; S., Banni; B., Botti; F. P., Corongiu; M. A., Dessì; IANNONE, Anna; VANNINI, Vanio; M. U., Dianzani
abstract

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.

1987 - Intracellular chromium reduction [Articolo su rivista]
P., Arslan; M., Beltrame; Tomasi, Aldo
abstract

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO2−4 through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4′-isothiocyanatostilbene-2-2′-disulfonic acid.

1987 - Spin trapping of free radical intermediates produced during the metabolism of isoniazid and iproniazid in isolated hepatocytes. [Articolo su rivista]
E., Albano; Tomasi, Aldo
abstract

By the use of spin trapping agents phenyl-t-butyl nitrone (PBN) and 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) free radical species were detected in isolated hepatocytes incubated with either isoniazid, iproniazid and their respective metabolites acetyl-hydrazine and isopropyl-hydrazine. The addition of bis-nitrophenyl phosphate, an inhibitor of the acylamidase enzymes, to isolated hepatocytes decreased the free radical activation of isoniazid and iproniazid, but not that of acetyl- and isopropylhydrazine, confirming that the radical species were originating from the biotransformation of these latter compounds. The ESR spectra were ascribed to the trapping of, respectively, acetyl and isopropyl free radicals on the basis of the analogies of the spectral feature with those of chemically-prepared spin adducts. Comparable ESR spectra were also observed during the metabolism of acetyl- and isopropylhydrazines by liver microsomes and their formation was inhibited by the omission of NADP+, anaerobic incubation and enzyme denaturation. In the microsomal preparations inhibitors of the mixed function oxidase system decreased to various extents the free radical formation and a similar effect was also observed following the destruction of cytochrome P-450 induced by pretreating the rats with CoCl2. The addition of reduced glutathione also decreased the radical trapping indicating that GSH can effectively compete with the spin traps for the reaction with the free radicals. The incubation of isolated hepatocytes with isoniazid or acetyl-hydrazine reduced by 20–25\% the intracellular GSH content, while a 50\% decrease in GSH was present in the cells exposed to iproniazid and isopropyl-hydrazine. In the same hepatocyte preparations stimulation of lipid peroxidation and leakage of LDH were also observed during cell incubation with iproniazid and isopropyl-hydrazine, but not with isoniazid or acetyl-hydrazine and the extent of both phenomena correlated with the susceptibility of the above compounds to the free radical activation

1986 - Decrease of hepatic mitochondrial glutathione and mitochondrial injury induced by 1,2-dibromoethane in the rat in vivo: Effect of diethylmaleate pretreatment [Articolo su rivista]
B., Botti; Bini, Anna; A., Calligaro; Meletti, Eros; Tomasi, Aldo; V., Vannini
abstract

Diethylmaleate (DEM) potentiated the 1,2-dibromoethane (DBE)-induced hepatic morphological lesion in fasted male Wistar rats, as revealed by light and electron microscopy examination. The subcellular structures involved in such lesions were the mitochondria. The potentiating effect of DEM appeared to be due to enhancement of the depletion of hepatic mitochondrial glutathione (GSH) caused by DBE. DEM, however, failed to potentiate the DBE-induced release in the plasma of hepatic enzymes. The relationship between loss of mitochondrial GSH, mitochondrial injury, and the importance of the mitochondrial lesion in DBE-induced hepatotoxicity is discussed.

1986 - Effect of spin traps in isolated rat hepatocytes and liver microsomes [Articolo su rivista]
E., Albano; K. H., Cheeseman; Tomasi, Aldo; R., Carini; M. U., Dianzani; T. F., Slater
abstract

Spin traps are increasingly employed in the detection of free radicals in biological systems, including liver microsomes and isolated hepatocytes. Two spin traps phenyl-t-butyl nitrone (PBN) and 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) have been tested for their effects on hepatocyte viability and mixed-function oxidase activity. High concentration of PBN but not of 4-POBN proved to moderately affect liver cell integrity, without interfering with intracellular ATP or cytochrome P-450 content. PBN also decreased hepatocyte GSH content, probably as the result of its metabolism to benzaldehyde. The two spin traps were found to inhibit aminopyrine demethylase and ethoxycoumarin deethylase activity in hepatocytes and microsomes. At low concentrations (1–5 mM) PBN enhanced aniline hydroxylase while high concentrations of the spin trap inhibited this activity. The inhibition of the monooxygenase system was not caused by damage of microsomal enzymes, but rather by competition with other substrates for the binding to the haemoprotein. The effects of spin traps on mixed function oxidase systems should be taken into account when evaluating the results of spin trapping experiments

1985 - Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique [Articolo su rivista]
Tomasi, Aldo; E., Albano; F., Biasi; T. F., Slater; V., Vannini; M. U., Dianzani
abstract

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 < CHBrCl2 < CHBr3 < CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihatomethanes might be of relevance to their established toxicity in the whole animal.

1985 - THE EFFECT OF MEDROXYPROGESTERONE ACETATE ON PREMATURE LABOR IN THE SHEEP [Articolo su rivista]
Tomasi, Aldo; H. Y., Tseng; A., Scommegna; L., Burd
abstract

The ability of medroxyprogesterone acetate to premature delivery delay was investigated in nine chronically catheterized pregnant sheep (125 to 130 days' gestation). Catheters were placed for measurement of intrauterine pressure and uterine vein concentrations of progesterone,17 beta-estradiol,and 15-keto-13,14-dihydroxyprostaglandin F2a. Premature parturition was induced in all animals by infusion of dexamethasone (1 mg/24 hours) to the fetus. Three ewes served as controls,three ewes received oral medroxyprogesterone acetate (5 mg/kg/day) 48 hours prior to the start of and during dexamethasone (early medroxyprogesterone acetate),and the remaining three animals received oral medroxyprogesterone acetate 12 hours after the onset of dexamethasone infusion (late medroxyprogesterone acetate). The lengths of time from dexamethasone until the onset of labor and from dexamethasone until delivery were compared,and were significantly longer for each group compared to control times. Of all endocrine events analyzed,the fall in plasma progesterone was the most consistent in all groups,showing that animals with rapid falls in progesterone levels (control group) tended to deliver earlier. These results demonstrate that medroxyprogesterone acetate can delay premature parturition in the sheep. Also medroxyprogesterone acetate appears to delay the endocrinologic events that normally occur at this time

1985 - The role of pentachlorophenol in causing mitochondrial derangement in hexachlorobenzene induced experimental porphyria [Articolo su rivista]
Masini, Alberto; D., Ceccarelli Stanzani; Tomasi, Aldo; T., Trenti; E., Ventura
abstract

Hexachlorobenzene feeding to rats for 60 days to induce experimental porphyria resulted in partial and constant uncoupling of oxidative phosphorylation of liver mitochondria from the early phase (i.e. 20 days) of treatment. Direct experimental evidence has been presented that this uncoupling is completely due to the action of pentachlorophenol endogenously formed by metabolism of hexachlorobenzene. The complete restoration of membrane potential by albumin under these conditions indicates that no irreversible damage occurs in the mitochondrial membrane. No appreciable correlation between concentrations of pentachlorophenol and the degree of porphyria has been observed

1984 - Detection of a free radical intermediate from divicine of vicia faba [Articolo su rivista]
E., Albano; Tomasi, Aldo; L., Mannuzzu; P., Arese
abstract

not available

1984 - STUDIES ON HUMAN UTERINE CERVIX AND RAT UTERUS USING S-BAND, X-BAND AND Q-BAND ELECTRON-SPIN-RESONANCE SPECTROSCOPY [Articolo su rivista]
Tomasi, Aldo; C., Benedetto; M., Nilges; F., Slater; H. M., Swartz; M. C. R., Symons
abstract

In previous studies we have reported on the detection of a strong e.s.r. signal in samples of normal human cervix; the signal is much reduced or absent in samples of invasive cancer of the cervix. In order to identify the species responsible for the strong signal, we have used X-, S- and Q-band e.s.r. spectroscopy. The major signal that is detectable in ground-up samples of cervix preserved at -196 degrees C has features consistent with the presence of a peroxy free radical. Good agreement with the experimental findings was obtained by computer simulation, using values for the g-tensor of gx = 2.002, gy = 2.005 and gz = 2.036. The peroxy radical is produced on grinding the normal cervix samples to a powder under liquid N2, and appears to be formed by modification of a pre-existing oxygen-containing complex. Control experiments eliminated the possibility that the strong signals seen in frozen powders prepared from normal cervix were artefacts only of the grinding procedure. Experiments with rats in vivo and with cervix samples in vitro are consistent with the conclusion that the peroxy radical is formed by disturbing the cyclo-oxygenase system that is involved in prostaglandin synthesis.

1984 - The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes [Articolo su rivista]
K., Cheeseman; E., Albano; Tomasi, Aldo; T., Slater
abstract

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 $\mu$mol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parellel with the decrease in microsomal enzyme activities.

1984 - Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation [Articolo su rivista]
E., Albano; G., Poli; Tomasi, Aldo; Bini, Anna; V., Vannini; M., Dianzani
abstract

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as $\alpha$-tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.

1983 - Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes [Articolo su rivista]
Tomasi, Aldo; Albano, Emanuele; M., Dianzani; T., Slater; Vannini, Vanio
abstract

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using 13C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.

1983 - The metabolism of halothane by hepatocytes: A comparison between free radical spin trapping and lipid peroxidation in relation to cell damage [Articolo su rivista]
Tomasi, Aldo; S., Billing; A., Garner; T., Slater; E., Albano
abstract

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.

1982 - Hydroxyl free radical production in iron-cysteine solutions and protection by zinc [Articolo su rivista]
A., Searle; Tomasi, Aldo
abstract

Hydroxyl radicals (OH−) can be formed on incubation of an oxygenated solution of ferrous sulphate and cysteine. This has been demonstrated by esr using the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide), catalase, and the radical scavengers ethanol and propan-2-ol. Hydroxyl radicals are not formed when excess zinc sulphate is present. These results provide support for the pro-oxidant action of iron and cysteine and a possible protective role for zinc.

1982 - SPIN-TRAPPING STUDIES ON THE FREE-RADICAL PRODUCTS FORMED BY METABOLIC-ACTIVATION OF CARBON-TETRACHLORIDE IN RAT-LIVER MICROSOMAL FRACTIONS-ISOLATED HEPATOCYTES AND INVIVO IN THE RAT [Articolo su rivista]
E., Albano; K. A. K., Lott; T. F., Slater; A., Stier; M. C. R., Symons; Tomasi, Aldo
abstract

The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM

1981 - ELECTRON-SPIN RESONANCE STUDIES ON NORMAL HUMAN-UTERUS AND CERVIX AND ON BENIGN AND MALIGNANT UTERINE-TUMORS [Articolo su rivista]
C., Benedetto; A., Bocci; M. U., Dianzani; B., Ghiringhello; T. F., Slater; Tomasi, Aldo; V., Vannini
abstract

Electron spin resonance (ESR) studies at −130° have been made on frozen samples of normal human cervix and uterus and on frozen samples of various pathological conditions of the cervix and uterus including fibroleiomyoma and carcinoma. Fifty-five samples of normal cervix and endometrium, 40 samples of nonmalignant disturbances, 15 benign tumor samples, and 20 malignant samples were studied. Very strong ESR signals were seen in frozen powders and frozen intact samples of normal cervix and endometrium and in nonmalignant gynecological conditions. In many cases, the ESR signal was greatly decreased or even undetectable in cancer samples. The substance(s) responsible for the ESR signal in frozen intact tissue (g = 2.11 to 2.15) is decreased in concentration when the sample is ground to powder under liquid nitrogen, and an anisotropic signal (g = 2.002 to 2.035) then becomes much more evident. The ESR signals in intact and in powder samples are sensitive to temperature variations; the signals disappear around 0°, and only the intact samples show significant recovery of signal on recooling. The anisotropic g values and temperature sensitivity in the powders may result from an organic peroxy radical that is more strongly associated with a metal ion in intact samples.

1980 - Spin trapping of free radical products of CCI4 activation using pulse radiolysis and high energy radiation procedures [Articolo su rivista]
Tomasi, Aldo; E., Albano; K., Lott; T., Slater
abstract

not available

1978 - MORPHOLOGICAL-CHANGES AND FREE-RADICAL RATE IN EARLY HEPATIC INJURY FROM CARBON-TETRACHLORIDE AND MONOBROMOTRICHLOROMETHANE [Articolo su rivista]
Bini, Anna; B., Botti; A., Calligaro; L., Congiu; Tomasi, Aldo; V., Vannini
abstract

not available