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Biomedical subjects

F Blasi

Publications and source records attributed to F Blasi.

At least 109 records · Page 6Linked to original sources
PubMed1998

Age at symptom onset and distribution by sex and symptoms in patients sensitized to different allergens.

The aim of our study was to analyze the clinical features, particularly the age at symptom onset, of allergic subjects (asthma and/or rhinitis) on the basis of the etiologic elements (sensitization to various allergens). We identified a group of monosensitized patients and a group of polysensitized patients. Within these groups, we identified subgroups of subjects monosensitized to one of the five main allergenic mixes (mites, Gramineae, trees, Parietaria, and Artemisia) and five subgroups of patients sensitized nonexclusively, that is, polysensitized, to the same allergens. The comparison between the two groups and among the various subgroups enabled us to conclude that: 1) mono- and polysensitized patients present some clinical features so different as to constitute two clearly distinct clinical groups 2) analysis of the clinical features associated with the sensitization to a specific allergen brings us to significantly different conclusions when we consider subgroups of monosensitized or polysensitized patients 3) the parameter "age at symptom onset" shows great heterogeneity among both the mono- and the polysensitized subgroups--in particular, the great differences in mean age among the monosensitized subgroups (trees>Artemisia>Parietaria>Gramineae>mites) appear very interesting and are open to various interpretative hypotheses 4) unlike the polysensitized group, in the monosensitized group and subgroups, mean age is similar between men and women and, only for tree- and Parietaria-monosensitive patients, also between asthmatic and rhinitic subjects.

Adolescent↗
PubMed1997

A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity.

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA-/- mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA-induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA-uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease-sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59(hck) tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.

Amino Acid Sequence↗
PubMed1997

Characterization of UEF-4, a DNA-binding protein required for transcriptional synergism between two AP-1 sites in the human urokinase enhancer.

The enhancer of the inducible urokinase gene depends on three essential but not sufficient transactivating elements, an upstream PEA3/AP-1A and a downstream AP-1B site. Enhancer activity also requires the interposed 74-base pair-long cooperation mediator (COM) region that allows transcriptional synergism between the transactivating sites. The 5'-half of COM (uCOM) forms four retarded complexes with HeLa or Hep-G2 nuclear proteins (UEF-1-4). We have identified the binding sequence for UEF-4 and generated uCOM elements uniquely mutated in the UEF-4-binding site or uniquely binding UEF-4. Introduction of these and other mutations in the context of the urokinase enhancer showed that all uCOM sites are important for enhancer activity but that UEF-4 and UEF-1 plus UEF-2/3 can substitute for each other, suggesting functional redundancy of urokinase enhancer factors. UEF-4 was purified from HeLa nuclear extract by affinity chromatography and shown to contain two polypeptides of 105 and 65 kDa, respectively, of which at least the former was endowed with DNA binding activity.

Base Sequence↗
PubMed1997

Glutamate-dependent activation of NF-kappaB during mouse cerebellum development.

NF-kappaB and activator protein 1 (AP-1) are dimeric transcription factors involved in transcriptional regulation in many cells, including neurons. We have examined their activity during mouse cerebellum development, a postnatal process starting just after birth and completed by the fourth postnatal (PN) week. The activity of these factors was analyzed by binding of nuclear extracts to a synthetic oligonucleotide representing the kappaB site of human immunodeficiency virus or the AP-1 site of the urokinase promoter. NF-kappaB activity was observed from 7 PN, was restricted to the developing cerebellum, and was not observed in the early postnatal neocortex and hippocampus. On the other hand, AP-1 activity was not found in cerebellum but was present in both neocortex and hippocampus. Moreover, a kappaB-driven transgene was found to be increasingly expressed in the cerebellum from 5 PN to 10 PN but not in the adult. The regulation of NF-kappaB activation in mouse cerebellum was analyzed by intraperitoneal injection of glutamate receptor antagonists to 9 PN mice, which abolished NF-kappaB-binding activity, suggesting an endogenous loop of glutamate receptor activation. Glutamate receptor agonists, on the other hand, induced NF-kappaB nuclear translocation in the cerebellum of 5 PN mice, which is a stage in which NF-kappaB is not yet endogenously activated. This effect was specific for NF-kappaB and not observed for AP-1. In adult mice, NF-kappaB activity was absent in the cerebellum and was not induced by intraperitoneal injection of glutamate receptor agonists. These data show that NF-kappaB is specifically activated during cerebellum development and indicate an important role of glutamate receptors in this process.

2-Amino-5-phosphonovalerate↗
PubMed1997

Nuclear levels of NF-kappaB correlate with syncytium-forming capacity of 8e51 cells, expressing a defective HIV virus.

The double NF-kappaB site identified in the LTR of the human immunodeficiency virus-1 (HIV-1) has been demonstrated to be necessary for efficient viral transcription. In this report we present the characterisation of NF-kappaB subunits engaged in complexes binding to the HIV-1 NF-kappaB site in human 8e51 T-cells, that harbour a defective HIV-1. At least four different specific NF-kappaB complexes are present in the nucleus of these cells. With the use of specific antibodies we have determined the composition of each complex using electrophoretic mobility shift assays. The results show the presence of several NF-kappaB family members, with the transactivating RelA being engaged in multiple complexes. The importance of NF-kappaB complexes in viral functions has been established comparing the level of NF-kappaB DNA-binding complexes with syncytia-forming activity of 8e51 cells. In fact, 8e51 cells that had almost lost their syncytia-forming capacity were found to contain at least 10 times less active NF-kappaB DNA-binding complex than the actively fusing cells. The correlation is specific as the level of at least three other transcription factors did not change.

Binding Sites↗
PubMed1997

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

Animals↗
PubMed1997

Through and beyond the wall: late steps in leukocyte transendothelial migration.

A combination of approaches has provided firm experimental support for a step-wise model of leukocyte adhesion to vascular endothelium under hemodynamic shear stress. However, leukocyte adhesion to vascular endothelium acquires functional significance only if coupled to transmigration and progression into extravascular tissues. As discussed here by Elisabetta Bianchi and colleagues, these latter processes involve the sequential activation of adhesion-dependent functional programs, carried out by structurally diverse multimolecular complexes.

Cell Adhesion↗
PubMed1997

Failure to detect the presence of Chlamydia pneumoniae in sarcoid pathology specimens.

The pathogenesis of sarcoidosis is not yet known. On the basis of seroepidemiological data, an association between Chlamydia pneumoniae infection and sarcoidosis has been suggested, but so far no study has addressed the direct detection of this agent in the affected tissues. The aim of the present study was to detect C. pneumoniae deoxyribonucleic acid (DNA) within sarcoid tissue specimens by means of a two-step polymerase chain reaction. Lung biopsy specimens of 33 patients with histologically confirmed pulmonary sarcoidosis and 21 control lung biopsies or pathology specimens of patients with pulmonary carcinoma or emphysema were retrospectively analysed. A nested polymerase chain reaction was applied using two sets of primers designed to detect a fragment of the 16 strand ribosomal ribonucleic acid (rRNA) gene of C. pneumoniae. The results of the study failed to demonstrate the presence of C. pneumoniae in biopsy specimens of sarcoid tissue and in the control lung biopsies or pathology specimens. Our results, therefore, tend to rule out the possibility of a direct involvement of Chlamydia pneumoniae in the pathogenesis of sarcoidosis.

Adult↗
PubMed1997

Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion.

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA/PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA/PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.

Antibodies↗
PubMed1997

A possible association of Chlamydia pneumoniae infection and acute myocardial infarction in patients younger than 65 years of age.

STUDY OBJECTIVES: We conducted a retrospective study on patients with acute myocardial infarction (AMI) and evaluated the incidence and prevalence of Chlamydia pneumoniae infection. METHODS: Sixty-one consecutive patients with AMI aged younger than 65 years were enrolled. Within 24 h of hospital admission, serum samples and pharyngeal swab specimens were obtained from all patients. In 49 of 61 patients, after a mean of 28 days from hospital admission, a second serum sample was drawn. A third serum sample was obtained in 23 of 61 patients. Serologic testing for Chlamydia pneumoniae was performed by a microimmunofluorescence test. We applied a nested-polymerase chain reaction for C pneumoniae DNA detection to pharyngeal swab specimens. Simultaneously, we performed a serologic study for C pneumoniae infection on 61 serum samples obtained from blood donors, matched for age, sex, and smoking habits. RESULTS: Serologic test results for C pneumoniae were consistent with acute reinfection in 12 patients, with chronic infection in 23 patients, and results were negative in 26 patients with AMI. In 3 of 12 patients with acute reinfection pattern and in 3 of 23 patients with chronic infection pattern, C pneumoniae DNA was detected on pharyngeal swab specimens. A significantly higher prevalence of IgG titers was observed in patients with AMI (35/61) compared to blood donors (18/61) (p=0.003). CONCLUSION: Our data confirm the possible role of C pneumoniae infection in coronary heart disease and suggest that reinfection may trigger the onset of AMI.

Antibodies, Bacterial↗
PubMed1996

Functional characterization of COM, a DNA region required for cooperation between AP-1 sites in urokinase gene transcription.

The inducible uPA enhancer contains two phorbol ester responsive elements: the combined PEA3/AP-1A and a downstream AP-1B site. The integrity of all three sites is essential for enhancer activity. The interposed 74 bp long DNA region (COM, Cooperation Mediator) is in turn required for the synergistic action of the PEA3/AP-1 and AP-1 sites. Here we present a characterization of the COM sequence: our results show that COM and COM-binding proteins (UEF, Urokinase Enhancer Factors) may play a structural role in the induction of the uPA enhancer by phorbol-myristate-acetate (TPA). (1) COM has a bipartite structure (uCOM and dCOM) and each half contributes by about 50% to the COM-mediated TPA induction. (2) COM function is strictly dependent on its position, but not on its orientation and neither the entire COM nor individual UEF-binding sites can directly transactivate a test promoter. (3) The requirement for COM in TPA-induction is partly eliminated by the deletion of COM sequence. However, also in the COM-deleted enhancer, integrity of the PEA3/AP-1A and AP-1B sites is essential for enhancer function. We conclude that COM and COM-binding proteins provide a structural surface which facilitates the cooperation between the transactivator proteins bound at the PEA3/AP-1A and AP-1B sites. Sequences homologous to uCOM are found in the promoters of other inducible genes, coding for proteases, cytokines and chemokines: for example, in the promoter of the MIP-1alpha/LD78 chemokine gene, a 15/18 nucleotides identity is found in a region mediating positive and negative functions in TPA induction. Thus, COM-like elements represent a general enhancer function which, although lacking an intrinsic transactivating capacity, modulates the synergism between transcription factors bound to distant sites.

Base Sequence↗
PubMed1996

NF-kappa B-mediated regulation of urokinase gene expression by PMA and TNF-alpha in human A549 cells.

Binding sites for different transcription factors have been identified in the regulatory region of the human uPA gene. We investigated the role of NF-kappa B and AP-1 families of transcription factors in the induction of uPA mRNA by TNF-alpha and PMA in the A549 cell line constitutively expressing uPA mRNA and protein. Using the protein synthesis inhibitor cycloheximide and the antioxidant PDTC, that have an opposite effect on NF-kappa B and AP-1 activation, we showed that uPA mRNA induction by TNF-alpha and PMA in the A549 cell line is mainly due to NF-kappa B activation.

Antioxidants↗
PubMed1996

Integrin-dependent induction of functional urokinase receptors in primary T lymphocytes.

In order to reach the sites of inflammation, lymphocytes leave the bloodstream and migrate into peripheral tissues, in a process involving integrin-mediated adhesion to the vascular endothelium, followed by transmigration across the endothelial barrier and through the underlying interstitial matrix. We have investigated the role of the plasminogen activator/plasmin system in normal T cell migration. Receptors for urokinase plasminogen activator (uPAR) were not expressed in resting T lymphocytes, but could be efficiently induced at the mRNA and protein level by coclustering of the antigen receptor complex and beta1 or beta2 integrins, through a signalling pathway involving both protein kinase C activation and an increase in intracellular cyclic AMP. Catalytic activation of plasminogen by uPAR-expressing T cells promoted their migration through an extracellular matrix in vitro. Plasmin-induced invasion was inhibited by plasmin-and urokinase inhibitors and by anti-uPAR antibodies. Finally, cytofluorimetric and immunohistochemical analysis of primary human tumor specimens showed the presence of uPAR positive infiltrating T cells in vivo. Collectively, these findings suggest that plasminogen activation may play a role in lymphocyte migration in vivo, and that integrin-dependent expression of membrane-associated endopeptidases could represent an additional step in the regulated process of leukocyte transmigration.

Adenylyl Cyclases↗
PubMed1996

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).

Animals↗