Paolo Paroncini - Academia.edu (original) (raw)

Papers by Paolo Paroncini

Environmental Microbiology Reports, Nov 25, 2013

To gain insights into the relationships and the genetic exchange among environmental and clinical... more To gain insights into the relationships and the genetic exchange among environmental and clinical enterococci, 59 strains (29 from marine aquaculture sites and 30 from clinical settings) resistant to tetracycline, erythromycin, ampicillin and/or gentamicin were analysed for the antibiotic resistance tet(M), tet(L), tet(O), erm(A), erm(B), mef blaZ, aac(6&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;)-Ie aph(2″)-Ia and virulence gelE, cylB, efaA and esp genes, and for the copper resistance gene tcrB. Antibiotic resistance and virulence genes were detected more frequently in clinical than in environmental enterococci; the opposite was true for copper resistance. Conjugation experiments demonstrated the transfer of antibiotic resistance genes from marine to clinical enterococci in interspecific mating and the uncommon joint transfer of tet(L) and erm(B). Enterobacterial repetitive intergenic consensus polymerase chain reaction typing evidenced a cluster (90% similarity) encompassing strains carrying multiple antibiotic resistance genes from both sets; the others marine isolates exhibited polyclonality and bore tcrB. Our results demonstrate that antibiotic-resistant marine enterococci bear antibiotic resistance genes transferable to humans and suggest that copper resistance, not observed among clinical strains, may be useful for survival in the environment, whereas virulence genes likely confer no advantage to enterococcal populations adapted to a lifestyle outside the host.

Enterococcal abundance in the farm sediments and at the control site was determined by q... more Enterococcal abundance in the farm sediments and at the control site was determined by qPCR before and after incubation with antibiotic-supplemented BHI broth. A, St. 1; B, St. 2 and C, St. 3. *Not detectable i.e.</p

Primer pairs used to detect resistance genes in PCR assays.</p

Journal of Biological Chemistry, 2005

Arthritis & Rheumatism, 2001

Objective. To explore the role of reactive oxygen species (ROS) in the in vitro activation of ski... more Objective. To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). Methods. Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O 2 ؊ and H 2 O 2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47 phox-specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3 H-thymidine incorporation. Northern blot analysis was used to study ␣1 and ␣2 type I collagen gene expression. Results. Unstimulated skin fibroblasts from SSc patients released more O 2 ؊ and H 2 O 2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47 phox , was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1␤ (IL-1␤), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor ␤1 (TGF␤1), and H 2 O 2. Treatment of normal and SSc fibroblasts with tumor necrosis factor ␣ (TNF␣), IL-2, IL-4, IL-6, IL-10, interferon-␣ (IFN␣), IFN␥, granulocyte-macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGF␤1, PDGF-BB, and other agonists (IL-4, IL-6, TNF␣, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of ␣1(I) and ␣2(I) collagen messenger RNA. Conclusion. The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase-like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O 2 ؊ , H 2 O 2 , IL-1␤, TGF␤1, PDGF-BB, IL-4, IL-6, TNF␣, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

Clinical and experimental rheumatology

PTX3 is a secreted molecule which consists of a C-terminal domain similar to classical pentraxins... more PTX3 is a secreted molecule which consists of a C-terminal domain similar to classical pentraxins (e.g. C-reactive protein) and of an unrelated N-terminal domain. Unlike the classical pentraxins, PTX3 is expressed in response to IL-1beta and TNF-alpha but not to IL-6. The present study was designed to investigate the expression of PTX3 in normal and scleroderma fibroblasts. Normal and SSc fibroblasts were cultured in the presence and absence of inflammatory cytokines. PTX3 mRNA expression in fibroblasts was evaluated by Northern analysis. PTX3 protein levels in fibroblast culture medium were estimated by ELISA. Normal fibroblasts were induced to express high levels of P7X3 mRNA by IL-1beta and TNF-alpha but not by other cytokines or growth factors. Scleroderma fibroblasts, unlike normal fibroblasts, constitutively expressed high levels of PTX3 in the absence of deliberate stimulation. The constitutive expression of PTX3 in SSc fibroblasts was not modified by anti-TNF-alpha antibodie...

Arthritis Research & Therapy, 2005

Chaperonins have classically been thought of as intracellular molecules involved in the correct f... more Chaperonins have classically been thought of as intracellular molecules involved in the correct folding of proteins. Their expression is upregulated during times of stress such as heat (hence their common nomenclature as heat shock proteins [HSP]), anoxia, hypoglycaemia and reactive oxygen species [1]. These are conditions found in infected tissues or in tissues with chronic inflammation such as the rheumatoid synovium. In their intracellular location they protect the cell from apoptotic death due to stress. Increasingly chaperonins have been recognised to subserve extracellular functions for which they have received the name 'chaperokines' since they bind to specific receptors on the cell surface and activate cells of the innate immune system to secrete inflammatory cytokines, chemokines and small molecular weight mediators such as prostaglandins [2]. Indeed, an early event in inflammation is cell stress/necrosis leading to the release of HSP60 and HSP70 that binds via a CD14-mediated mechanism to Toll-like receptors 2 and 4 [2] as part of the 'danger' signal [3]. The secretion of tumour necrosis factor alpha, IL-1, IL-12 and other chemokines prepares the environment for a TH1 adaptive immune response. It is now recognised that some chaperonins, such as BiP and HSP27, may activate the innate immune system to secrete anti-inflammatory cytokines, such as IL-10 [4,5] that may skew the adaptive immune response to TH2. Recent work by our group has shown that BiP can not only prevent but also treat ongoing collagen-induced arthritis in DBA/1 mice [6], suggesting that chaperonins may down modulate ongoing TH1 responses. Thus, it may be possible to suppress rheumatoid inflammation by administration of appropriate chaperonins such as BiP. Finally, chaperonins may be important system regulators determining the outcome between TH1 and Th2 immune responses. References 1. Pockley AG: Heat shock proteins as regulators of the immune response. Lancet 2003, 362:469-476. 2. Asea A: Chaperokine-induced signal transduction pathways. Exerc Immunol Rev 2003, 9:25-33. 3. Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305. 4. De AK, Kodys KM, Yeh BS, Miller-Graziano C: Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heatshock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus.

aAmerican Type Culture Collection.</p

*detected both in sediment and in broth.</p

PLoS ONE, 2013

Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic e... more Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic environment through release of large amounts of chemicals, including antibiotics. Moreover, the presence of organic matter and bacteria of different origin can favor gene transfer and recombination. Whereas the consequences of such activities on environmental microbiota are well explored, little is known of their effects on allochthonous and potentially pathogenic bacteria, such as enterococci. Sediments from three sampling stations (two inside and one outside) collected in a fish farm in the Adriatic Sea were examined for enterococcal abundance and antibiotic resistance traits using the membrane filter technique and an improved quantitative PCR. Strains were tested for susceptibility to tetracycline, erythromycin, ampicillin and gentamicin; samples were directly screened for selected tetracycline [tet(M), tet(L), tet(O)] and macrolide [erm(A), erm(B) and mef] resistance genes by newly-developed multiplex PCRs. The abundance of benthic enterococci was higher inside than outside the farm. All isolates were susceptible to the four antimicrobials tested, although direct PCR evidenced tet(M) and tet(L) in sediment samples from all stations. Direct multiplex PCR of sediment samples cultured in rich broth supplemented with antibiotic (tetracycline, erythromycin, ampicillin or gentamicin) highlighted changes in resistance gene profiles, with amplification of previously undetected tet(O), erm(B) and mef genes and an increase in benthic enterococcal abundance after incubation in the presence of ampicillin and gentamicin. Despite being limited to a single farm, these data indicate that aquaculture may influence the abundance and spread of benthic enterococci and that farm sediments can be reservoirs of dormant antibiotic-resistant bacteria, including enterococci, which can rapidly revive in presence of new inputs of organic matter. This reservoir may constitute an underestimated health risk and deserves further investigation.

Environmental Microbiology Reports, 2013

Environmental Science & Technology, 2013

Fecal indicator bacteria (FIB) are used worldwide to assess water quality in coastal environments... more Fecal indicator bacteria (FIB) are used worldwide to assess water quality in coastal environments, but little is known about their genetic diversity and pathogenicity. This study examines the prevalence, antimicrobial resistance, virulence, and genetic diversity of FIB isolated from marine sediments from a central Adriatic seaside resort. FIB, recovered from 6 out of 7 sites, were significantly more abundant at sampling stations 300 m offshore than close to the shore. Escherichia coli accounted for 34.5% of fecal coliforms, and Enterococcus faecalis accounted for 32% of enterococci. Most isolates (27% of E. coli and 22% of enterococci) were recovered from the sediments that had the highest organic content. Multidrug-resistant E. coli (31%) and enterococci (22%) were found at nearly all sites, whereas 34.5% of E. coli and 28% of enterococci harboring multiple virulence factors were recovered from just two sites. Pulsed-field gel electrophoresis typing showed wide genetic diversity among isolates. Human epidemic clones ( E. coli ST131 and Enterococcus faecium ST17) were identified for the first time by multilocus sequence typing in an area where bathing had not been prohibited. These clones were from sites far removed from riverine inputs, suggesting a wide diffusion of pathogenic FIB in the coastal environment and a high public health risk.

Journal of Biological Chemistry, 2003

Journal of Biological Chemistry, Jan 16, 2003

We have characterized the role of c-Myb and B-Myb in the regulation of human type I collagen ␣2 c... more We have characterized the role of c-Myb and B-Myb in the regulation of human type I collagen ␣2 chain gene expression in fibroblastic cells. We have identified four Myb-binding sites (MBSs) in the promoter. Transactivation assays on wild type and mutant promoter-reporter constructs demonstrated that c-Myb, but not B-Myb, can transactivate the human type I collagen ␣2 chain gene promoter via the MBS-containing region. Electrophoretic mobility shift assay experiments showed that c-Myb specifically binds to each of the four MBS; however, the mutagenesis of site MBS-4 completely inhibited transactivation by c-Myb, at least in the full-length promoter. In agreement with these results, c-myb ؊/؊ mouse embryo fibroblasts (MEFs) showed a selective lack of expression of type I collagen ␣2 chain gene but maintained the expression of fibronectin and type III collagen. Furthermore, transforming growth factor-␤ induced type I collagen ␣2 chain gene expression in c-myb ؊/؊ MEFs, implying that the transforming growth factor-␤ signaling pathway is maintained and that the absence of COL1A2 gene expression in c-myb ؊/؊ MEFs is a direct consequence of the lack of c-Myb. The demonstration of the importance of c-Myb in the regulation of the type I collagen ␣2 chain gene suggests that uncontrolled expression of c-Myb could be an underlying mechanism in the pathogenesis of several fibrotic disorders.