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Biomedical subjects

P Sebo

Publications and source records attributed to P Sebo.

At least 19 recordsLinked to original sources
PubMed2006

[Preparing a drug list for primary care].

To limit drug adverse effects, the use of a limited choice of drugs is desirable. We identified 29 frequent health problems and selected first and second choice medication based on the following criteria: clinical efficacy based on medical evidence or expert consensus, safety profile, and costs. For each substance, adverse effect, contraindication, interaction risk, specific dosing, and safety use during pregnancy and lactation were reviewed. More than seventy substances were identified. This list is available for download at the following address (in French): http://www.hcuge.

Formularies, Hospital as Topic↗
PubMed2006

Aeromonas bacteremia in an elderly immunocompetent patient.

We report the case of an elderly immunocompetent patient with Aeromonas hydrophila bacteremia without evidence of portal of entry. Despite several risk factors for a poor outcome, such as impaired renal function, two positive blood cultures, and community-acquired infections, the patient survived. Antimicrobial susceptibility was normal. Unknown polycystic liver disease was discovered and misdiagnosed as a hepatic abscess at the time of the bacteremia which was confirmed by repeated CT scans. Because of the absence of other risk factors for Aeromonas bacteremia, hepatic polycystic disease may take part in the onset of Aeromonas sp bacteremia as well as immunosenescence.

Aeromonas↗
PubMed2005

[Treating hypertension: a challenge for the general practitioner].

Hypertension is associated with a greater risk of developing a stroke or a coronary heart disease, but remains, for the meanwhile, undertreated in a vast majority of patients. Non pharmacological interventions can reduce blood pressure and should be recommended to every patient suffering from hypertension. Nevertheless, a drug therapy must often be introduced. The threshold at which a treatment should be started depends mostly on the cardiovascular risk of each patient and the benefit of antihypertensive drug therapy depends primarily on the reduction of the blood pressure itself.

Antihypertensive Agents↗
PubMed2001

Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase. Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC.

The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.

Acylation↗
PubMed2001

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9.

The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Adenylate Cyclase Toxin↗
PubMed2001

Neisseria meningitidis RTX protein FrpC induces high levels of serum antibodies during invasive disease: polymorphism of frpC alleles and purification of recombinant FrpC.

The Neisseria meningitidis FAM20 strain secretes two proteins of unknown function, FrpA and FrpC, which contain typical RTX domains found in cytotoxins from other gram-negative pathogens. To evaluate whether the Frp proteins could be involved in meningococcal virulence, 65 isolates of all serogroups were screened by PCR for the presence of both frp genes. The frpA allele was, however, poorly conserved. A single strain harbored an frpA allele of the previously described size, while large insertions were detected in the frpA loci of 22 isolates (34%), and the 42 remaining isolates (65%) did not contain frpA at all. In contrast, frpC alleles, albeit of variable length, were detected in all invasive and most carrier strains. This suggests that meningococci may produce a family of FrpC proteins of various molecular masses. High levels of both immunoglobulin G (IgG) and IgA class antibodies recognizing recombinant FrpC were indeed detected in convalescent-phase sera of most patients at 2 and 4 to 5 weeks after the first symptoms of meningococcal disease. These results show that FrpC-like proteins are produced and may play a role in invasive meningococcal infections.

Alleles↗
PubMed2001

Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity.

CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8(+) T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8(+) CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.

Adenylyl Cyclases↗
PubMed2000

Occurrence of IgA and IgG autoantibodies to calreticulin in coeliac disease and various autoimmune diseases.

Calreticulin (CRT), a high-affintiy calcium binding protein and chaperone, was recently identified as one of the targets of autoantibodies in coeliac disease. We evaluated the level of IgA and IgG antibodies to CRT in sera from patients with coeliac disease and various autoimmune diseases. The level of antibodies to gliadin (shown previously to cross-react with CTR), isolated enterocytes and tissue transglutaminase were determined for comparison. The mean level of IgA antibodies to CRT was significantly higher (P< 0.001) in sera from coeliac patients with active disease (139.9+/-11.2 AU/+/-SE) than in healthy controls (20.9+/-1.7 AU). In sera of patients with systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), multiple sclerosis (MS) and autoimmune thyroiditis (AT) or inflammatory bowel disease (IBD) the mean level (25.8+/-3.7 to 38.1+/-5.6 AU) did not exceed the cut-off value. A low level of these antibodies, however, was detected in some sera of patients with MS and IBD. The level of IgG anti-CRT antibodies was increased in coeliac patients (mean 125.4+/-8.0 AU, P< 0.001) when compared to that in healthy controls (33.9+/-2.3 AU). The IgG anti-CRT antibodies were also detected in about 30% of SLE patients sera (54.1+/-3.6 AU, P< 0.001), but the mean level reached only half that detected in coeliac patients.

Adolescent↗
PubMed2000

Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites.

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.

Adenylate Cyclase Toxin↗
PubMed2000

Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis.

The adenylate cyclase (CyaA) of Bordetella pertussis delivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8(+) T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8(+) T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8(+) CD4(+) T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.

Adenylate Cyclase Toxin↗
PubMed1999

An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels.

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.

Adenylate Cyclase Toxin↗
PubMed1999

The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status.

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.

Acylation↗
PubMed1999

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying multiple copies of a viral CD8(+) T-cell epitope.

Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.

Adenylate Cyclase Toxin↗
PubMed1999

Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.

Adenylate Cyclase Toxin↗
PubMed1997

Anti-viral protection conferred by recombinant adenylate cyclase toxins from Bordetella pertussis carrying a CD8+ T cell epitope from lymphocytic choriomeningitis virus.

The elucidation of the mechanisms of antigen presentation by major histocompatibility complex class I molecules has stimulated the search for nonreplicative vectors that could deliver CD8+ T cell epitopes to the cytosol of antigen-presenting cells to trigger the activation of specific cytotoxic T lymphocytes (CTLs) in vivo. In the present study, we investigated the potential ability of an invasive adenylate cyclase toxin from Bordetella pertussis, carrying a CD8+ T cell epitope from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), to stimulate protective anti-viral immunity. Mice immunized with this recombinant toxin developed strong CTL responses against LCMV-infected target cells. Moreover, these mice were protected against an intracerebral challenge with a virulent strain of LCMV that killed all nonimmunized mice within 7 days. This protection was abolished after in vivo elimination of CD8+ T cells. A mutant toxin devoid of adenylate cyclase activity (i.e., cAMP synthesizing activity) was constructed by insertion of a dipeptide into the catalytic site of the molecule. This genetically detoxified invasive toxin carrying the LCMV epitope stimulated a strong CTL response against both peptide-coated and virus-infected target cells, and mice immunized with this molecule were fully protected against a lethal intracerebral LCMV challenge. To our knowledge, this study represents the first demonstration that a genetically detoxified bacterial toxin carrying a viral CTL epitope can stimulate efficient protective immunity.

Adenylyl Cyclases↗
PubMed1996

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8+ T cell epitopes.

Exogenous Ags enter the endosomal pathway and are presented to CD4+ T cells in association with class II molecules whereas endogenously synthesized Ags, such as viral proteins, are presented to CD8+ T cells in association with MHC class I molecules. Therefore, most CTL activation strategies use live vectors although an alternative possibility could be to deliver the epitope into the cytosol by targeting it to an invasive nonreplicative vector. The adenylate cyclase toxin of Bordetella pertussis is able to invade a large number of eukaryotic cells and to deliver its catalytic domain to the cytosol of the cells. In the present study, we have tested the in vivo immunogenicity of recombinant adenylate cyclase toxins expressing CTL epitopes either from the nucleoprotein of lymphocytic choriomeningitis virus or from the V3 region of HIV-1 gp120. BALB/c mice immunized with these toxins developed high specific CTL responses that were shown to be mediated by class I-restricted CD8+ CTL. The induction of CTL responses by recombinant toxins did not require CD4+ T cells and the cytotoxic activity persisted 2 mo after immunization. The activation of CTL responses by the recombinant adenylate cyclase toxin required the full-length invasive activity of the toxin but did not depend upon its catalytic adenylate cyclase activity as demonstrated with a genetically inactivated recombinant toxin expressing the lymphocytic choriomeningitis virus epitope. This genetically detoxified invasive toxin represents, therefore, an attractive new vector for CTL activation.

Adenylyl Cyclases↗
PubMed1995

Interaction of calcium with Bordetella pertussis adenylate cyclase toxin. Characterization of multiple calcium-binding sites and calcium-induced conformational changes.

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity. Binding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (KD = 0.5-0.8 mM) Ca2+ binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca(2+)-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.

Adenylate Cyclase Toxin↗