Anna Bardoni

Analysis of bronchoalveolar lavage fluid proteome from systemic sclerosis patients with or without functional, clinical and radiological signs of lung fibrosis

Lung fibrosis is a major cause of mortality and morbidity in systemic sclerosis (SSc). However, its pathogenesis still needs to be elucidated. We examined whether the alteration of certain proteins in bronchoalveolar lavage fluid (BALF) might have a protective or a causative role in the lung fibrogenesis process. For this purpose we compared the BALF protein profile obtained from nine SSc patients with lung fibrosis (SScFib+) with that obtained from six SSc patients without pulmonary fibrosis (SScFib-) by two-dimensional gel electrophoresis (2-DE). Only spots and spot-trains that were consistently expressed in a different way in the two study groups were taken into consideration. In total, 47 spots and spot-trains, corresponding to 30 previously identified proteins in human BALF, showed no significant variation between SScFib+ patients and SScFib- patients, whereas 24 spots showed a reproducible significant variation in the two study groups. These latter spots corresponded to 11 proteins or protein fragments, including serum albumin fragments (13 spots), 5 previously recognized proteins (7 spots), and 4 proteins (3 spots) that had not been previously described in human BALF maps, namely calumenin, cytohesin-2, cystatin SN, and mitochondrial DNA topoisomerase 1 (mtDNA TOP1). Mass analysis did not determine one protein-spot. The two study groups revealed a significant difference in BALF protein composition. Whereas levels of glutathione S-transferase P (GSTP), Cu–Zn superoxide dismutase (SOD) and cystatin SN were downregulated in SScFib+ patients compared with SScFib- patients, we observed a significant upregulation of α1-acid glycoprotein, haptoglobin-α chain, calgranulin (Cal) B, cytohesin-2, calumenin, and mtDNA TOP1 in SScFib+ patients. Some of these proteins (GSTP, Cu–Zn SOD, and cystatin SN) seem to be involved in mechanisms that protect lungs against injury or inflammation, whereas others (Cal B, cytohesin-2, and calumenin) seem to be involved in mechanisms that drive lung fibrogenesis. Even if the 2-DE analysis of BALF did not provide an exhaustive identification of all BALF proteins, especially those of low molecular mass, it allows the identification of proteins that might have a role in lung fibrogenesis. Further longitudinal studies on larger cohorts of patients will be necessary to assess their usefulness as predictive markers of disease.

Differential expression of β1,3galactosyltransferases in human colon cells derived from adenocarcinomas or normal mucosa1

Two β1,3galactosyltransferases are detected in human colon cells: one corresponds to β3GalT1, the other (β3GalTx) is found to be different from any cloned β3GalT since in vitro it utilizes GlcNAc very efficiently under specific reaction conditions. Expression of β3GalT1 transcript is high in normal colon mucosa and control neuroectodermal cells, which do not express sialyl‐Lewis a antigen, and low in colon adenocarcinoma cells, as assessed by competitive RT‐PCR. β3GalTx activity is high in adenocarcinoma cells expressing sialyl‐Lewis a and undetectable in all other cells, suggesting differential involvement and opposite regulation of such enzymes during carcinogenesis.

2-DE and LC-MS/MS for a comparative proteomic analysis of BALf from subjects with different subsets of inflammatory myopathies.

The protein profiles of bronchoalveolar lavage fluid (BALf) of patients belonging to three selected subsets of Polymyositis/Dermatomyositis (PM/DM) have been compared by using a combination of 2-DE and MALDI-TOF/MS or LC-MS/MS. Our study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype. From among the 323+/-51 protein spots that may represent the most highly expressed proteins in BALf of these patients, 24 unique spots were isolated and proteins identified. In particular, 9 spots were present in BALf of PM/DM patients only; 12 spots were exclusive of Overlap patients and 3 spots of AS patients. From among the proteins identified, a few were classified as cytoskeletal proteins, others were involved in oxidative stress and a number of proteins were associated with general metabolic activity or immunological response and inflammation. This is the first study in which evidence is provided that a number of different proteins are expressed in different subsets of PM/DM and supports our contention that the proteomic approach would be beneficial in discovering molecules which could represent possible prognostic factors of these rare pathologies.

Proteomic analysis of lymphoblastoid cells from Nasu-Hakola patients: a step forward in our understanding of this neurodegenerative disorder.

Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer’s disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses.

Recent novel MEKC applications to analyze free amino acids in different biomatrices: 2009-2010.

This report intends to provide an updated overview of the most important methodological developments of MEKC in the field of qualitative/quantitative analysis of free amino acids in different matrices. A good number of articles published in the years 2009–2010 addresses the main applications of such procedures together with their advantages and/or drawbacks. The usefulness of chiral CE selective methods for the separation of D‐amino acids in biological and food samples and the use of microchips, as well as other foreseen trends in different areas, are also discussed.

Recent applications of CE- and HPLC-MS in the analysis of human fluids

The present review intends to cover the literature on the use of CE‐/LC‐MS for the analysis of human fluids, from 2010 until present. It has been planned to provide an overview of the most recent practical applications of these techniques to less extensively used human body fluids, including, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate, tear fluid, breast fluid, amniotic fluid, and cerumen. Potential pitfalls related to fluid collection and sample preparation, with particular attention to sample clean‐up procedures, and methods of analysis, from the research laboratory to a clinical setting will also be addressed. While being apparent that proteomics/metabolomics represent the most prominent approaches for global identification/quantification of putative biomarkers for a variety of human diseases, evidence is also provided of the suitability of these sophisticated techniques for the detection of heterogeneous components carried by these fluids.

Indoleamine 2,3-Dioxygenase in Lung Allograft Tolerance

BACKGROUND Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the degradation of tryptophan (Try) to kynurenine (Kyn), is thought to suppress T-cell activity. Although a few experimental studies have suggested a role for IDO in graft acceptance, human data are scarce and inconclusive. We sought to establish whether, in lung transplant recipients (LTRs), plasma IDO activity mirrors the level of graft acceptance. METHODS We measured the plasma Kyn/Try ratio, reflecting IDO activity, by high-performance liquid chromatography (HPLC) in 90 LTRs, including 26 patients who were still functionally/clinically stable for >36 post-transplant months (stable LTRs) and 64 LTRs with bronchiolitis obliterans syndrome (BOS, Grades 0-p to 3). Twenty-four normal healthy controls (NHCs) were also included. RESULTS The Kyn/Try ratio in stable LTRs resembled that observed in NHCs, whereas, unexpectedly, patients with BOS, who had lower counts of peripheral CD4(+) T-regulatory cells and tolerogenic plasmacytoid dendritic cells than stable LTRs, showed an increased plasma Kyn/Try ratio compared with both NHCs and stable LTRs. IDO expression by in vitro-stimulated peripheral blood mononuclear cells (PBMC) did not vary between BOS and stable LTRs. Furthermore, BOS patients displayed signs of chronic systemic inflammation (increased plasma levels of interleukin-8 and tumor necrosis factor-alpha) and higher T-cell activation (increased frequency of peripheral interferon-gamma-producing clones). CONCLUSIONS Our results suggest that, in vivo, in lung transplantation, plasma IDO activity does not reflect the degree of lung graft acceptance, but instead is correlated with the degree of chronic inflammation.

Ixodes ricinus and Its Endosymbiont Midichloria mitochondrii: A Comparative Proteomic Analysis of Salivary Glands and Ovaries

Hard ticks are hematophagous arthropods that act as vectors of numerous pathogenic microorganisms of high relevance in human and veterinary medicine. Ixodes ricinus is one of the most important tick species in Europe, due to its role of vector of pathogenic bacteria such as Borrelia burgdorferi and Anaplasma phagocytophilum, of viruses such as tick borne encephalitis virus and of protozoans as Babesia spp. In addition to these pathogens, I. ricinus harbors a symbiotic bacterium, Midichloria mitochondrii. This is the dominant bacteria associated to I. ricinus, but its biological role is not yet understood. Most M. mitochondrii symbionts are localized in the tick ovaries, and they are transmitted to the progeny. M. mitochondrii bacteria have however also been detected in the salivary glands and saliva of I. ricinus, as well as in the blood of vertebrate hosts of the tick, prompting the hypothesis of an infectious role of this bacterium. To investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, we generated the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, we were able to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method here developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii.

Proliferation characteristics and polyploidization of cultured myofibroblasts from a patient with fibroblastic rheumatism

Fibroblast-like cells were obtained from a nodule of a patient with fibroblastic rheumatism, and grown in culture for different times (from passage 3 to 21). These cells as well as the fibroblasts taken from an unaffected skin area (controls) of the same patient, have been investigated by fluorescence microscopy, cytochemical methods and cytometry, to evaluate their cytodifferentiation features and cytokinetic characteristics. In addition, in low-passage cultures, the secretion of collagen and of non-collagenic proteins was evaluated using electrophoretic techniques. The immunolabeling with antibodies against sm-specific a-actin (which was taken as a marker of myofibroblasts) showed that, already in low-passage cultures, the percentage of myofibroblasts was higher in the nodule-derived cell populations, and progressively increased with increasing passages. This suggests that myofibroblasts have higher proliferation potential than control fibroblasts. Myofibroblasts were also found to undergo polyploidization and hypertrophy, especially in high-passage cultures. Based on these results, it may be hypothesized that in fibroblastic rheumatism the development of the typical nodules could depend on the intrinsic capability of myofibroblats of proliferating faster than normal fibroblasts and of becoming polyploid and hypertrophic. Nodule-derived cells in culture synthesized slightly less collagen and non-collagen proteins than did the control fibroblasts; this suggests that the increased fibrosis observed in nodules in situ could be likely dependent on a reduced degradation of the extracellular matrix components.

Antinuclear antibodies in systemic sclerosis

Antinuclear autoantibodies are present in more than 90% of patients with systemic sclerosis (SSc). Some of these antibodies can be regarded as disease-specific: anticentromere antibodies, anti-topoisomerase I and anti-RNA polymerase I–III identify the three major serological subsets. Other important antibodies recognize nucleolar antigens, such as fibrillarin, Th/To and PM/Scl. All of these specific serological reactivities are mutually exclusive, have definite clinical associations and prognostic implications. This makes antinuclear antibodies a valuable tool in classifying and managing patients with SSc. SSc pathogenesis remains unclear and the role of autoantibodies is debated. Studies exploring their relationships with new emerging disease mechanisms are still in progress.