Biomedical subjects

Francesco Blasi

Publications and source records attributed to Francesco Blasi.

15 recordsLinked to original sources

PubMed2003

The role of urokinase-type plasminogen activator (uPA)/uPA receptor in HIV-1 infection.

The binding of urokinase-type plasminogen activator (uPA) to its glycosyl-phosphatidyl-inositol (GPI) anchored receptor (uPAR) mediates a variety of functions in terms of vascular homeostasis, inflammation and tissue repair. Both uPA and uPAR, as well as their soluble forms detectable in plasma and other body fluids, represent markers of cancer development and metastasis, and they have been recently described as predictors of human immunodeficiency virus (HIV) disease progression, independent of CD4+ T cell counts and viremia. A direct link between the uPA/uPAR system and HIV infection was earlier proposed in terms of cleavage of gp120 envelope by uPA. More recently, a negative regulatory effect on both acutely and chronically infected cells has been linked to the noncatalytic portion of uPA, also referred to as the amino-terminal fragment (ATF). ATF has also been described as a major CD8+ T cell soluble HIV suppressor factor. In chronically infected promonocytic U1 cells this inhibitory effect is exerted at the very late stages of the virus life cycle, involving virion budding and entrapment in intracytoplasmic vacuoles, whereas its mechanism of action in acutely infected cells remains to be defined. Since uPAR is a GPI-anchored receptor it requires association with a signaling-transducing component and different partners, which include CD11b/CD18 integrin and a G-protein coupled receptor homologous to that for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine. Which signaling coreceptor(s) is(are) responsible for uPA-dependent anti-HIV effect remains currently undefined.

Chemokines↗

PubMed2003

Overexpression of PREP-1 in F9 teratocarcinoma cells leads to a functionally relevant increase of PBX-2 by preventing its degradation.

To bind DNA and to be retained in the nucleus, PBX proteins must form heterodimeric complexes with members of the MEINOX family. Therefore the balance between PBX and MEINOX must be an important regulatory feature. We show that overexpression of PREP-1 influences the level of PBX-2 protein maintaining the PREP-1-PBX balance. This effect has important functional consequences. F9 teratocarcinoma cells stably transfected with PREP-1 had an increased DNA binding activity to a PREP-PBX-responsive element. Because PREP-1 binds DNA efficiently only when dimerized to PBX, the increased DNA binding activity suggests that the level of PBX might also have increased. Indeed PREP-1-overexpressing cells had a higher level of PBX-2 and PBX-1b proteins. PBX-2 increase did not depend on increased mRNA level or a higher rate of translation but rather because of a protein stabilization process. Indeed, PBX-2 level drastically decreased after 3 h of cycloheximide treatment in control but not in PREP-1-overexpressing cells and the proteasome inhibitor MG132 prevented PBX-2 decay in control cells. Hence, dimerization with PREP-1 appears to decrease proteasomal degradation of PBX-2. Retinoic acid induces differentiation of F9 teratocarcinoma cells with a cascade synthesis of HOX proteins. In PREP-1-overexpressing cells, HOXb1 induction was more sustained (3 days versus 1 day) and the induced level of MEIS-1b, another TALE (three amino acid loop extension) protein involved in embryonal development, was higher. Thus an increase in PREP-1 leads to changes in the fate-determining HOXb1 and has therefore important functional consequences.

Amino Acid Sequence↗

PubMed2003

Detection of Chlamydiae pneumoniae but not Helicobacter pylori DNA in atherosclerosis plaques.

Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylori infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques.

Aged↗

PubMed2003

Therapeutic potential of the new quinolones in the treatment of lower respiratory tract infections.

The impact of respiratory infections on public health is increasing and lower respiratory tract infections are a major cause of morbidity and mortality. We are also facing a worldwide burst of antibiotic bacterial resistance. The new fluoroquinolones have an excellent spectrum covering the most important respiratory pathogens, including atypical and 'typical' pathogens. Pharmacokinetic and dynamic properties of the new fluoroquinolones have a significant impact on their clinical and bacteriological efficacy. They cause a concentration-dependent killing with a sustained postantibiotic effect. Fluoroquinolones combine exceptional efficacy with cost-effectiveness. Not surprisingly, different guidelines have inserted these agents among the drugs of choice in the empirical therapy of community-acquired pneumonia. This review discusses the more recent data on bacteriological and clinical activity and critically analyses the risks of a potential overuse of this valuable new class of drugs.

4-Quinolones↗

PubMed2002

Characteristics of Streptococcus pneumoniae and atypical bacterial infections in children 2-5 years of age with community-acquired pneumonia.

The characteristics of community-acquired pneumonia associated with Streptococcus pneumoniae infection were compared with those associated with atypical bacterial infection and with mixed S. pneumoniae-atypical bacterial infection in 196 children aged 2-5 years. S. pneumoniae infections were diagnosed in 48 patients (24.5%); atypical bacterial infections, in 46 (23.5%); and mixed infections, in 16 (8.2%). Although white blood cell counts and C-reactive protein levels were higher in patients with pneumococcal infections, no other clinical, laboratory, or radiographic characteristic was significantly correlated with the different etiologic diagnoses. There was no significant difference in the efficacy of the different treatment regimens followed by children with S. pneumoniae infection, whereas clinical failure occurred significantly more frequently among children with atypical bacterial or mixed infection who were not treated with a macrolide. This study shows the major role of both S. pneumoniae and atypical bacteria in the development of community-acquired pneumonia in young children, the limited role of clinical, laboratory, and radiological features in predicting etiology, and the importance of the use of adequate antimicrobial agents for treatment.

Child, Preschool↗

PubMed2002

Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells.

Activated transcription of the urokinase-type plasminogen activator (uPA) gene depends on the enhancer, located approximately 2 kb from the start of transcription. The proximal promoter, driving basal transcription, contains a GC-/GA-rich sequence immediately upstream of the TATA box. We have investigated the role played by this element in the transcription of the uPA gene in HeLa and PC3 cells, which do not express or constitutively express the gene, respectively. This region binds either Sp1 or Sp3, as monomers or multimers, but not a combination of the 2 proteins. The more efficient binding of Sp1 to the proximal promoter in PC3 cells is correlated to its phosphorylation state. Polymerase chain reaction (PCR)-coupled, chromatin immunoprecipitation experiments with anti-Sp1 antibodies indeed show an enrichment of proximal promoter sequences in PC3 cells and support the observed difference in transcription levels from proximal promoter constructs in HeLa versus PC3 cells. Furthermore, overexpression of Sp1 increases transcription from the reporter construct in HeLa cells, whereas in PC3 cells, overexpression of Sp3 does not reduce transcription from the same construct, indicating that the Sp1/Sp3 balance cannot be shifted. We conclude that the GC-/GA-rich element of the uPA regulatory region is an independent functional element, regulated by Sp family proteins. Phosphorylation of Sp1 determines the presence in vivo and the functionality of this element in PC3 cells. Thus, the cellular context determines the relevance of the GC-/GA-rich region in uPA gene transcription, which contributes to constitutive gene expression, related, in turn, to the invasive phenotype.

Adenocarcinoma↗

PubMed2002

Urokinase-urokinase receptor interaction mediates an inhibitory signal for HIV-1 replication.

Elevated levels of soluble urokinase-type plasminogen activator (uPA) receptor, CD87/u-PAR, predict survival in individuals infected with HIV-1. Here, we report that pro-uPA (or uPA) inhibits HIV-1 expression in U937-derived chronically infected promonocytic U1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-alpha (TNF-alpha). However, pro-uPA did not inhibit PMA or TNF-alpha-dependent activation of nuclear factor-kB or activation protein-1 in U1 cells. Cell-associated HIV protein synthesis also was not decreased by pro-uPA, although the release of virion-associated reverse transcriptase activity was substantially inhibited, suggesting a functional analogy between pro-uPA and the antiviral effects of IFNs. Indeed, cell disruption reversed the inhibitory effect of pro-uPA on activated U1 cells, and ultrastructural analysis confirmed that virions were preferentially retained within cell vacuoles in pro-uPA treated cells. Neither expression of endogenous IFNs nor activation of the IFN-inducible Janus kinase/signal transducer and activator of transcription pathway were induced by pro-uPA. Pro-uPA also inhibited acute HIV replication in monocyte-derived macrophages and activated peripheral blood mononuclear cells, although with great inter-donor variability. However, pro-uPA inhibited HIV replication in acutely infected promonocytic U937 cells and in ex vivo cultures of lymphoid tissue infected in vitro. Because these effects occurred at concentrations substantially lower than those affecting thrombolysis, pro-uPA may represent a previously uncharacterized class of antiviral agents mimicking IFNs in their inhibitory effects on HIV expression and replication.

Electrophoretic Mobility Shift Assay↗

PubMed2002

Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization.

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.

Animals↗

PubMed2002

uPAR: a versatile signalling orchestrator.

The plasminogen system has been implicated in clot lysis, wound healing, tissue regeneration, cancer and many other processes that affect health and disease. The urokinase receptor uPAR was originally thought to assist the directional invasion of migrating cells, but it is now becoming increasingly evident that this proteinase receptor elicits a plethora of cellular responses that include cellular adhesion, differentiation, proliferation and migration in a non-proteolytic fashion.

Animals↗

PubMed2002

Reliability of nested PCR for detection of Chlamydia pneumoniae DNA in atheromas: results from a multicenter study applying standardized protocols.

The present multicenter study was designed to find explanations for the discrepancies in the reported rates of detection of Chlamydia pneumoniae DNA in endarterectomy specimens. Coded identical sets of (i) a C. pneumoniae DNA dilution series (panel 1; n = 10), (ii) spiked control tissue specimens (panel 2; n = 10 specimens, including 5 negative controls), and (iii) endarterectomy specimens (panel 3; 15 atheromas, 5 negative controls) were analyzed at four laboratories by three standardized DNA extraction methods in each laboratory and a nested touchdown PCR protocol targeting the ompA gene of C. pneumoniae. Panel 1 samples were correctly identified as positive to levels of 0.3 inclusion-forming units (IFU)/PCR mixture (100%) and 0.03 IFU/PCR mixture (50%). All negative controls were correctly reported as negative. Panel 2 samples were identified as C. pneumoniae positive to levels of 0.01 IFU/PCR mixture (100%) and 0.005 IFU/PCR mixture (91%), independent of the DNA extraction method used, and only one false-positive result was reported. For panel 3 samples, 5 of 240 (2%) analyses (in which DNA extractions and PCR were performed at the same laboratory) were positive; the positive specimens were from three endarterectomy specimens and two negative controls. After exchange of DNA extracts between laboratories, 13 of 15 atheroma samples were C. pneumoniae DNA positive in at least 1 of a series of 48 analyses per atheroma sample; however, the overall positivity rate did not exceed 5% (33 of 720 analyses) and therefore was lower than that for the negative controls (8%; 19 of 240 analyses). Not a single positive result could be achieved when all panel 3 extracts (n = 240 analyses) were reamplified by a 16S rRNA PCR followed by hybridization with a C. pneumoniae-specific probe. Statistical analyses demonstrated that positive results did not occur in an independent and random fashion and could most likely be explained by amplicon carryover at the nested PCR level as well as amplicon introduction during DNA extraction, but not by the patterns of distribution of very low target levels or a certain DNA extraction protocol. The results of studies by nested PCR for detection of the prevalence of C. pneumoniae will always be questionable.

Arteriosclerosis↗

PubMed2002

Therapies in development for community-acquired pneumonia.

The current use of antimicrobials has become more complex due to the extensive emergence of antibiotic resistance. The single most important approach in resistance control is probably the judicious use of chemotherapeutic agents. New agents that may be of use in the treatment of community-acquired pneumonia are currently in development. Antimicrobials can be grouped according to their mechanism of action. These include protein synthesis inhibitors (ketolides, oxazolidinones, streptogramins and glycylcyclines), nucleic acid synthesis inhibitors (fluoroquinolones), peptidoglycan synthesis inhibitors (beta-lactams and glycopeptides) and agents interfering with membrane function (cationic peptides and lipopeptides). Among those agents under development, only the oxazolidinones, the cationic peptides and the lipopeptide antibiotics can be truly regarded as structurally novel inhibitors as the other agents are analogues of existing compounds which have been in clinical use for many years.

Anti-Bacterial Agents↗

PubMed2002

Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor.

Proteolytic cleavage of the urokinase plasminogen activator receptor (uPA(R)) prevents the binding of uPA and vitronectin while generating biologically active uPAR fragments. We have recently shown that matrix metalloproteinase-12 (MMP-12) releases cellular uPAR-antigen from stimulated human micro-vascular endothelial cells providing a novel feedback mechanism between the plasminogen activation and MMP systems. We now show that MMP-12 and other MMPs directly and efficiently cleave uPAR at the Thr86 paralal Tyr87 peptide bond located in the linker region connecting uPAR domains 1 and 2, releasing the major ligand binding domain 1 from the rest of the receptor. The possible biological importance of uPAR cleavage by MMPs is supported by the observation that also murine uPAR is cleaved by MMP-12 (at the Pro89 paralal Gln90 peptide bond), despite the limited sequence homology between the linker regions. Using an antibody raised against the human uPAR linker region we show that this region of uPAR, which contains the chemotactic SRSRY epitope, is exposed upon MMP cleavage.

Amino Acid Motifs↗

PubMed2000

Chlamydia pneumoniae respiratory infections.

Chlamydia pneumoniae is a significant cause of both upper and lower respiratory tract infections. The spectrum of diseases ranges from asymptomatic infection to serious disease, including severe pneumonia and exacerbations of chronic bronchitis requiring mechanical ventilation. There is increasing evidence of involvement of C. pneumoniae infection in bronchial asthma, and the role of this agent in immunocompromised patients has also begun to be appreciated.

Journal Article↗

PubMed1996

Clinical features of Chlamydia pneumoniae acute respiratory infection.

Chlamydia pneumoniae is a worldwide respiratory pathogen involved in 6--20% of community-acquired pneumonias and in about 5% of acute exacerbations of chronic bronchitis. Preliminary data also indicate a possible association between Chlamydia pneumoniae infection and asthma. Further studies are needed to elucidate whether Chlamydia pneumoniae is merely a precipitant of asthma symptoms or is actually one of the causes of asthma.

Journal Article↗

PubMedDate not supplied

The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy.

Cancer dissemination and metastasis is synonymous with invasive cell migration; a process in which the extracellular matrix (ECM) plays the dual role of the substratum on which the cells move as well as the physical obstacle that the cells have to surpass. To degrade the physical obstacle, which the ECM poses in the direction of migration, cells use proteolytic enzymes capable of degrading the ECM components. A major protease system responsible for ECM degradation is the plasminogen activation system, which generates the potent serine protease plasmin. The subject of this review, the urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plays an impressive range of distinct, but overlapping functions in the process of cancer invasion and metastasis: Firstly, uPA/uPAR promotes extracellular proteolysis by regulating plasminogen activation. Secondly, uPA/uPAR regulates cell/ECM interactions as an adhesion receptor for vitronectin (Vn) and through its capacity to modulate integrin function. Thirdly, uPA/uPAR regulates cell migration as a signal transduction molecule and by its intrinsic chemotactic activity. This review is focused on recent insight into the cancer related biology of the uPA/uPAR system as well as its implications for clinical cancer diagnosis, prognosis and therapy.

Animals↗