Riccardo Cianti

Protein profile of capacitated versus ejaculated human sperm.

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs during sperm transport through the female genital tract, and it is physiologically not complete until the spermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by using appropriate capacitation media. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. This work deals with a proteomic approach to the analysis of protein profile variations in human normospermic samples as a consequence of three hours in vitro capacitation. 2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitative and qualitative significant variations were found. Among the MS obtained identifications, proteins with a significant decrease after capacitation were found to be involved in protein fate, metabolism, and flagellar organization; on the contrary, increasing proteins were found to be related to cellular stress. Interestingly, the detected flagellar organization proteins decreased during capacitation whereas their corresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopulation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observed in the entire sperm population. An immunofluorescence analysis of this sperm typology was carried out with antiactin and antitubulin antibodies.

Transketolase and 2′,3′-Cyclic-nucleotide 3′-Phosphodiesterase Type I Isoforms Are Specifically Recognized by IgG Autoantibodies in Multiple Sclerosis Patients

The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin β4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2′,3′-cyclic-nucleotide 3′-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications.

Calgranulin B (S100A9) Levels in Bronchoalveolar Lavage Fluid of Patients with Interstitial Lung Diseases

Calgranulins are small calcium-binding proteins with several immunological functions involved in inflammatory processes. Calgranulin A is reported to be mainly associated with acute inflammation while calgranulin B seems to play a role in chronic inflammatory disorders. In this study we used a proteomic approach to analyse calgranulin B expression in bronchoalveolar lavage (BAL) from a group of patients with different interstitial lung diseases. Two dimensional electrophoresis analysis of BAL was performed in 11 idiopathic pulmonary fibrosis patients, nine sarcoidosis patients, 11 with systemic sclerosis patients and five healthy controls. Significantly higher (p < 0.001) calgranulin B percentage volumes were observed in BAL from IPF patients than controls and other ILD patients. This result sustains the hypothesis that calgranulin B could be involved in chronic lung diseases, probably through increased expression and enhanced activation of alveolar polymorphonuclear cells related to idiopathic pulmonary fibrosis. Quantitative analysis by an easier method applied to a larger population will be necessary to determine whether calgranulin B could be a good marker of disease severity.

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short‐ (1 injection) and long‐term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short‐term and one long‐term MPE treatment. One group received EV alone. Two test groups received EV with either a short‐term or long‐term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2‐DE/MALDI‐TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV‐induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.

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

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

Using peripheral blood mononuclear cells to determine proteome profiles in human cardiac failure.

In chronic heart failure (CHF), peripheral blood mononuclear cells (PBMC) might undergo structural and/or functional alterations as a consequence of the development and progression of the disease.