High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics

<p>Abstract</p> <p>Background</p> <p>Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels.</p> <p>Results</p> <p>Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis) indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm) we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS).</p> <p>Conclusion</p> <p>This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.</p

Studio della S-nitrosilazione in vitro e in vivo in Arabidopsis thaliana

L\u2019ossido nitrico (NO) \ue8 una molecola segnale di importanza centrale in numerosi processi cellulari sia negli animali sia nelle piante. In particolare l\u2019NO agisce nelle piante sia come regolatore dello sviluppo in quanto coinvolto in processi fisiologici come la germinazione, la crescita di foglie e radici, la senescenza e la fioritura, sia come segnale chiave nella risposta difesa contro i patogeni. Tuttavia pur essendo noti i numerosi processi fisiologici in cui l\u2019NO \ue8 coinvolto, ancora non risultano chiari quali siano i suoi specifici \u201ctarget\u201d molecolari. La S-nitrosilazione consiste nel legame di un gruppo NO al tiolo libero di una cisteina. Nei sistemi animali questa modificazione \ue8 risultata essere responsabili dell\u2019alterazione, attraverso cambiamenti della struttura e della conformazione, dell\u2019attivit\ue0 di proteine coinvolte in processi cruciali della cellula, quali l\u2019apoptosi. Inoltre essendo molto specifica l\u2019S-nitrosilazione potrebbe rappresentare un meccanismo di controllo e di trasduzione del segnale durante particolari condizioni della cellula specie in relazione alle risposte stress a ossidativo e nitrosativo. In questo lavoro di tesi \ue8 stato messo a punto un saggio per l\u2019analisi dell\u2019Snitrosilazione in pianta. Come riferimento \ue8 stato preso il metodo \u201cbiotin switch\u201d (Jaffrey e coll., 2001) che permette di convertire le proteine S-nitrosilate in proteine biotinilate, in modo da riconoscerle attraverso il loro legame specifico all\u2019anticorpo anti-biotina o di isolarle tramite cromatografia di affinit\ue0. Il protocollo \ue8 stato adattato alla rilevazione e all\u2019isolamento delle proteine S-nitrosilate in pianta, seguita da analisi tramite tecniche di elettroforesi bidimensionale. Il metodo \ue8 stato quindi utilizzato per identificare le proteine di A. thaliana S-nitrosilate in vitro e quelle S-nitrosilate in vivo durante la risposta ipersensibile (HR), meccanismo di difesa della pianta dai patogeni avirulenti che porta ad una morte cellulare programmata nella zona d\u2019infezione. Trattando estratti proteici di A. thaliana con agente nitrosilante, nello specifico Snitrosoglutatione (GSNO) ed effettuando il \u201cbiotin switch\u201d seguito da analisi in gel bidimensionali \ue8 stato possibile identificare, tramite spettrometria di massa MALDITOF, 55 proteine S-nitrosilate. Tra queste proteine sono stati ritrovati enzimi del sistema antiossidante, proteine coinvolte nella detossificazione di sostanze tossiche e nella regolazione dello stato redox, proteine coinvolte nella risposta a stress biotici e abiotici. Il sistema utilizzato \ue8 stato quindi applicato all\u2019analisi delle proteine S-nitrosilate di A. thaliana differenzialmente espresse durante la HR. L\u2019analisi delle proteine soggette a S-nitrosilazione in pianta dopo l\u2019infezione del patogeno avirulento Pst AvrB ha portato all\u2019identificazione, tramite spettrometria di massa MALDI-TOF, di 18 proteine tra cui la perossiredossina II E (Prx II), enzima coinvolto nella detossificazione dell\u2019H2O2. L\u2019S-nitrosilazione della Prx II E \ue8 stata verificata in vivo e in vitro dopo trattamento con GSNO; inoltre \ue8 stato verificato che la modificazione post-traduzionale, che avviene 8h dopo l\u2019infezione del patogeno avirulento, riguarda l\u2019intero \u201cpool\u201d di proteina presente durante la HR.Non disponibil

Cross Talk between Reactive Nitrogen and Oxygen Species during the Hypersensitive Disease Resistance Response

Review relativa ai ruoli di ossido nitrico e specie reattive dell'ossigeno nella morte cellulare ipersensibile delle piante, come forma estrema di resistenza a patogeni

Free light chains and heavy/light chains in monitoring POEMS patients.

Background: POEMS syndrome is defined by Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy and Skin changes. The vascular endothelial growth factor (VEGF) appears to play a key role in the pathogenesis of the syndrome, and its concentrations are deemed to correlate to disease activity. The aim of the present study was to verify whether other biochemical markers including serum free light chains (FLC) and heavy/light chains (HLC) would be of value in monitoring POEMS patients. Methods: Fifty-three serum samples were collected from seven POEMS patients at diagnosis and during a follow-up period (range 14-56 months). VEGF was measured using an ELISA method, while FLC and HLC concentrations were measured using Binding Site reagents on a BNII (Siemens) nephelometer. Results: At diagnosis all patients presented high VEGF concentrations, while the \u3ba/\u3bbFLC ratio (FLCr) was within the reference range. Four patients had abnormal HLC, HLC\u3ba/HLC\u3bb (HLCr) and FLC values. The relationship between the trend of VEGF and both HLC and FLC during the follow-up was analysed by means of Cohen's \u3ba coefficient. VEGF and HLC values displayed a significant \u3ba-Cohen (0.537, p=0.002) in all chemotherapy-responder patients while in non-responders it did not. Conversely, in both responders and non-responders, VEGF and FLC values did not attain a significance on \u3ba-Cohen analysis. In three out of four responders HLCr values increased, thus reflecting an improved clinical condition. Conclusions: The findings made in the present study indicate that HLC, either as intact immunoglobulin or as HLCr, may provide useful information, particularly in identifying responders and confirm that the role of FLC is unreliable in monitoring patients with POEMS syndrome

Evaluation of hepcidin isoforms in hemodialysis patients by a proteomic approach based on SELDI-TOF MS

The hepatic iron regulator hormone hepcidin consists, in its mature form, of 25 amino acids, but two other isoforms, hepcidin-20 and hepcidin-22, have been reported, whose biological meaning remains poorly understood. We evaluated hepcidin isoforms in sera from 57 control and 54 chronic haemodialysis patients using a quantitative proteomic approach based on SELDI-TOF-MS. Patients had elevated serum levels of both hepcidin-25 and hepcidin-20 as compared to controls (geometric means: 7.52 versus 4.69 nM, and 4.06 versus 1.76 nM, resp., P < .05 for both). The clearance effects of a single dialysis session by different dialysis techniques and membranes were also investigated, showing an average reduction by 51.3% +/- 29.2% for hepcidin-25 and 34.2% +/- 28.4% for hepcidin-20 but only minor differences among the different dialysis modalities. Measurement of hepcidin isoforms through MS-based techniques can be a useful tool for better understanding of their biological role in hemodialysis patients and other clinical condition

Hospitalization trends, costs, and risk factors in HIV-infected children on antiretroviral therapy.

OBJECTIVE: To assess hospitalization trends in HIV-infected children on antiretroviral therapy (ART) in Thailand, an important indicator of morbidity, ART effectiveness, and health service utilization. DESIGN: Prospective observational cohort METHOD: Children initiating ART in 1999-2009 were followed in 40 public hospitals. Hospitalization rate per 100 person-years were calculated from ART initiation to last follow-up/death. Costs to the healthcare provider were calculated using WHO inpatient estimates for Thailand. Zero-inflated Poisson models were used to examine risk factors for early (<12 months of ART) and late hospitalization (≥12 months) and frequency of admissions. RESULTS: A total of 578 children initiated ART, median follow-up being 64 months [interquartile range (IQR) 43-82]; 211 (37%) children were hospitalized with 451 admissions. Hospitalization rates declined from 63 per 100 person-years at less than 6 months to approximately 10 per 100 person-years after 2 years of ART, and costs fell from $35 per patient-month to under$5, respectively. Age less than 2 years, US Centers of Disease Control and Prevention stage B/C, and stunting at ART initiation were associated with early hospitalization. Among those hospitalized, baseline CD4 cell percentage less than 5%, wasting, initiation on dual therapy, late calendar year, and female sex were associated with higher incidence of early admissions (P <0.02). There were no predictors of late hospitalization, although previous hospitalization in less than 12 months of ART was associated with three times higher incidence of late admissions (P < 0.0001). CONCLUSION: One in three children required hospitalization after ART. Admissions were highest in the first year of therapy and rapidly declined thereafter. Young age, advanced disease stage, and stunting at baseline were predictive of early hospitalization. Treatment initiation before disease progression would likely reduce hospitalization and alleviate demands on healthcare services