Barbara Magi

Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis : a two-dimensional electrophoretic study

We used two‐dimensional (2‐D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty‐eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests. Comparison of the BALF protein maps of the two groups of patients showed 32 spots with statistically significant disease‐related variations in relative abundance. In sarcoidosis we found an increase in the amount of several plasma proteins, while in idiopathic pulmonary fibrosis we observed a statistically significant increase in low‐molecular‐weight proteins, many of which are involved in inflammatory processes (such as MIF and calgranulin) or antioxidant response (such as antioxidant peroxysomal enzyme and thioredoxin peroxidase 2). 2‐D electrophoresis allowed us to identify new BALF proteins and to characterize protein composition in patients with sarcoidosis and idiophatic pulmonary fibrosis. Comparison of the gels of the two diseases showed that they differ in BALF protein profiles as they do in type of immune response.

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation

AbstractWe previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, β-actin, tubulin-α-1-chain and enolase-α. Interestingly, all carbonylated proteins except calreticulin and enolase-α showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

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.

Nonprotein-bound iron and plasma protein oxidative stress at birth.

We previously reported plasma nonprotein-bound iron (NPBI) as a reliable early indicator of intrauterine oxidative stress (OS) and brain injury. We tested the hypothesis that albumin, an NPBI serum carrier, is the major target of NPBI-induced OS. Twenty-four babies were randomly selected from 384 newborns constituting the final cohort of a prospective study undertaken to evaluate the predictive role of NPBI in cord blood for neurodevelopmental outcome. Twelve were selected in the group with lowest NPBI levels (0–1.16 μM) and good neurodevelopmental outcome and 12 in the group with highest NPBI levels (≥15.2 μM) and poor neurodevelopmental outcome. Protein carbonyl groups were identified in cord blood samples by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting with anti-2,4-dinitrophenyl (DNP) antibodies. Two series of immunoreactive spots, corresponding to serum albumin and α-fetoprotein, were found only in the group with highest NPBI levels. We found an association between NPBI and carbonylated proteins in babies with highest NPBI levels. Since NPBI may produce hydroxyl radicals through the Fenton reaction, the major target of OS induced by NPBI is its carrier: albumin. Oxidation of albumin can be expected to decrease plasma antioxidant defenses and increase the likelihood of tissue damage due to OS in the newborns.

Cryptosporidium infection: diagnostic techniques

The aim of this study was to compare a rapid immunological test and a PCR method with the conventional morphological technique for the identification of Cryptosporidium in faecal samples. Cryptosporidium was found in five samples by Kinyoun acid-fast stain. Five samples yielded positive results on immunoassay, three of which yielded negative results on microscopy. Thus, only two patients were positive for Cryptosporidium according to both methods. PCR analysis confirmed only one sample as positive. Non-homogeneous distribution of parasites in stool samples, lack of oocysts in the tested sample and antigenic diversity among Cryptosporidium species may explain the poor agreement among the three tests. Based on our experience, microscopy test with Kinyoun stain is the best and cheapest way to detect Cryptosporidium spp. in faecal samples. With this method, we have found a 5.4% prevalence of Cryptosporidium infection in our area, similar to those reported for other regions of Italy and Europe.

Proteomic dissection of dome formation in a mammary cell line: Role of tropomyosin-5b and maspin

In this work we extended the study of genes controlling the formation of specific differentiation structures called “domes” formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the β-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC β-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC β-subunit and the rat8 proteins, and is under the negative control of maspin.

Presence of Macrophage Migration Inhibitory Factor in Human Milk: Evidence in the Aqueous Phase and Milk Fat Globules

Human milk is a source of bioactive substances regulating the development and activity of the newborn immune system. Human milk has been found to contain a number of cytokines, including interleukins, growth factors, and colony stimulating factors. In the present study, we assessed 10 specimens of human milk for the presence of macrophage migration inhibitory factor (MIF), a cytokine recently described in several human reproductive organs and tissues. Using biochemical as well as immunologic techniques, we showed that MIF is abundantly present in human milk, mostly distributed in the lipid layer and in the aqueous phase. Fractionation of the lipid layer showed that MIF is highly concentrated inside milk fat globules. In view of its proinflammatory features, we speculate that milk MIF may protect the newborn against infection and play a role in preserving the functionality of the lactating mammary gland. Furthermore, the localization of MIF in lipid globules suggests a possible strategy for the protection of milk cytokines from the gastric barrier.

Analysis of macrophage migration inhibitory factor (MIF) in patients with idiopathic pulmonary fibrosis.

By proteomic approach we previously characterised bronchoalveolar lavage (BAL) protein profiles of patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis and systemic sclerosis. Among differently expressed proteins we identified macrophage migration inhibitory factor (MIF), a multi-function pleiotropic cytokine. This study was performed to validate our findings by a further proteomic approach and ELISA in a larger population of patients and controls. MIF expression in lung tissue was also evaluated by immunohistochemistry. MIF was identified in all 2-DE gels of IPF patients and it was significantly increased compared to controls (p<0.05). This result was confirmed by ELISA: MIF concentrations were significantly higher in IPF patients than controls (p<0.001) and were directly correlated with neutrophil percentages (p=0.0095). Immunohistochemical analysis revealed enhanced expression in bronchiolar epithelium, alveolar epithelium, and fibroblastic foci. In conclusion, MIF is a pleiotropic cytokine that could be involved in the pathogenesis of IPF, being particularly abundant in BAL of these patients and mainly expressed in the areas of active fibrosis.

Proteome analysis of bronchoalveolar lavage in pulmonary langerhans cell histiocytosis

BackgroundPulmonary Langerhans-cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by clusters of Langerhans cells, organized in granulomas, in the walls of distal bronchioles. It is a diffuse lung disease related to tobacco smoking but otherwise of unknown etiopathogenesis.MethodsIn this study we used a proteomic approach to analyze BAL protein composition of patients with PLCH and of healthy smoker and non-smoker controls to obtain insights into the pathogenetic mechanisms of the disease, to study the effect of cigarette smoking on susceptibility to PLCH and to identify potential new biomarkers.ResultsTwo-dimensional electrophoresis and image analysis revealed proteins that were differently expressed (quantitatively and qualitatively) in the three groups of subjects. The proteins were identified by mass spectrometry and have various functions (antioxidant, proinflammatory, antiprotease) and origins (plasma, locally produced, etc.). Many, such as protease inhibitors (human serpin B3) and antioxidant proteins (glutathione peroxidase and thioredoxin) are already linked to PLCH pathogenesis, whereas other proteins have never been associated with the disease. Interestingly, numerous proteolytic fragments of plasma proteins (including kininogen-1 N fragments and haptoglobin) were also identified and suggest increased proteolytic activity in this inflammatory lung disease. Differences in protein expression were found between the three groups and confirmed by Principal Component Analysis (PCA).ConclusionAnalysis of BAL proteomes of PLCH patients and of smoker and non-smoker controls also proved to be useful for researching the pathogenetic mechanisms and for identifying biomarkers of this rare diffuse lung disease.

Evolutionary trends of neurofilament proteins in fish

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.