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Biomedical subjects

P Berche

Publications and source records attributed to P Berche.

At least 145 records · Page 8Linked to original sources
PubMed1991

[Antibiotic treatment of cystic fibrosis].

Lower respiratory tract superinfection is nearly constant in cystic fibrosis and has a significant impact on mortality. The three organisms which most often colonize the bronchial tree are Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa. The latter organism is found in 70% to 90% of older cystic fibrosis patients. P. aeruginosa infections are remarkably persistent and cause severe, extensive lung damage. Antimicrobial therapy is indicated in patients with clinical symptoms (fever, weight loss, changes in sputum and auscultation) and should be selected on the basis of cytobacteriologic studies of sputum. Oral antibiotics are usually successful in eradicating S. aureus and H. influenzae. Conversely, intravenous therapy is required in most cases of P. aeruginosa infection. Fifteen-day courses are given repeatedly, either on a routine basis every three months, or whenever new clinical symptoms develop. Antimicrobials usually fail to eradicate P. aeruginosa even when significant clinical improvement occurs. Two-drug therapy and judicious use of the various available anti-microbial agents should delay development of resistant strains, an event which is nearly inevitable as antimicrobial treatments are repeated.

Anti-Bacterial Agents↗
PubMed1991

T cell-mediated immunity to Cowdria ruminantium in mice: the protective role of Lyt-2+ T cells.

The inability of athymic nude mice to make a drug-aided recovery from infection with either the Kümm or the Welgevonden stocks of Cowdria ruminantium and their inability to mount an immune response, suggest that immunity in heartwater is cell-mediated. The adoptive transfer of immunity with the spleen cells of mice immune to the Welgevonden stock is supportive evidence. Immune spleen cells depleted of Lyt-2+ T cells are unable to confer resistance to challenge to recipient mice, whereas the depletion of L3T4+ T cells had no effect on the protection conferred by immune spleen cells. This is conclusive evidence that immunity in heartwater is largely cell-mediated. Immune serum, C. ruminantium and complement incubated in the presence of mouse peritoneal macrophages, inhibits the infectivity of the heartwater agent, but not in the absence of macrophages. The decreased resistance to challenge of immune mice treated with gloxazone adds further support to the concept that in heartwater persistence of C. ruminantium in the host is associated with immunity.

Animals↗
PubMed1990

Detection of anti-listeriolysin O for serodiagnosis of human listeriosis.

To see whether detection of antibodies against listeriolysin O (LLO) could be used to diagnose human listeriosis, sera from 28 patients infected with Listeria monocytogenes and 101 controls were tested by dot-blot titration with purified LLO. 27 patients (96.4%) with listeriosis produced specific anti-LLO. Anti-LLO was detected in 8 (15.6%) of 51 healthy controls and in 6 (12.0%) of 50 controls who had various bacterial, fungal, and viral infections. Anti-LLO titres did not exceed 100 in these two control groups. Anti-LLO could be detected soon after clinical onset of listeriosis, and antibodies persisted for at least several months. This test might be useful for epidemiological surveys and for serodiagnosis of listeriosis, especially when bacteria cannot be isolated.

Adolescent↗
PubMed1990

Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O.

Listeriolysin O (LLO), a major virulence factor of the intracellular bacterium Listeria monocytogenes, shares with other known 'thiol-activated toxins' a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon-induced non-haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full-length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys-484 to Ala and of 80% for mutation Cys-484 to Ser. Mutations Trp-491 and Trp-492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supernatants. These results demonstrate that the thiol group of Cys-484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp-492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non-haemolytic mutant Trp-492-Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains.

Alleles↗
PubMed1990

Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid.

Electron microscopic evidence is presented that bacteria harboring the virulence plasmid pYV from Yersinia pseudotuberculosis are localized in extracellular sites during the course of infection in mice, often unambiguously undergoing active replication. Virulent pYV+ bacteria, often seen adherent to platelets, severely restricted granuloma formation, creating necrotic microabscesses poorly populated with inflammatory cells. This contrasts with granulomas produced by pYV- bacteria, which appear to be composed mainly of polymorphonuclear and mononuclear cells. Our results therefore strongly suggest that active replication of pYV+ bacteria predominantly, if not exclusively, occurs in vivo in extracellular sites.

Extracellular Space↗
PubMed1989

Impact of genetically regulated T cell proliferation on acquired resistance to Listeria monocytogenes.

Two lines of mice genetically selected for high and low in vitro responses to PHA were used to evaluate the impact of T cell polyclonal expansion on acquired resistance to Listeria monocytogenes. The selective breeding induced two major consequences in low responder mice: (1) a reduction of the number of L3T4+ cells and (2) a restriction of T cell expansion upon PHA stimulation, predominantly affecting the Lyt-2+ subset, and associated with an abridgment of IL-2 production. In vivo PHA stimulation induced anti-Listeria protection in high responder mice, but was much less effective in low responder mice. Flow cytometer analysis revealed that T cell proliferation was also reduced in low responder mice during the course of Listeria infection, implying both L3T4+ and Lyt-2+ subsets. This defect did not apparently influence the kinetics of bacterial elimination in host tissues, which was similar in both lines during primary Listeria infection. In contrast, the expression of delayed-type hypersensitivity to Listeria antigens and the level of immunologic memory were significantly reduced in low responder mice. In vivo selective T cell depletion by anti-L3T4 or anti-Lyt-2 mAb allowed us to demonstrate the predominant role of Lyt-2+ cells in protection and that of L3T4+ cells in the expression of delayed-type hypersensitivity.

Animals↗
PubMed1989

Production of thiol-dependent haemolysins by Listeria monocytogenes and related species.

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.

Hemolysin Proteins↗
PubMed1989

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.

Animals↗
PubMed1989

Loss of catalase activity in Tn1545-induced mutants does not reduce growth of Listeria monocytogenes in vivo.

Two catalase-negative mutants of Listeria monocytogenes were obtained by chromosomal insertions of the conjugative transposon Tn1545. The loss of catalase activity did not reduce the level of virulence of these mutants in mice. Indeed, both mutants were capable of growing in host tissues at the same rate as the parental catalase-positive strain. These results favor the view that catalase does not play a critical role in the resistance of L. monocytogenes to macrophage killing.

Animals↗
PubMed1989

Reacquisition of virulence of haemolysin-negative Listeria monocytogenes mutants by complementation with a plasmid carrying the hlyA gene.

An haemolysin negative mutant of Listeria monocytogenes LO28 produced by insertion of Tn917 resulted in an avirulent derivative. The hlyA gene of the same strain was previously cloned in Escherichia coli and retransfered by transformation to the hly- derivative, after subcloning in the shuttle vector pMK4. The transformant strain (L828) reacquired an haemolytic activity at a similar level than the wild strain. The virulence of this hly+ transformant was estimated by determining the LD50 in Swiss mice infected intravenously. With increasing doses of bacteria a hly- control strain (transformant with only pMK4) appeared to be totally avirulent; however, no significant difference in virulence was found between the hly+ transformants and the wild strain. Seriol viable counts in the liver and spleen of infected mice demonstrated an increase in number of L828 hly+ transformants at 48 h, but the hly- control transformants were rapidly eliminated. These results confirm that the production of haemolysin is a major factor in the pathogenic capabilities of L. monocytogenes.

Bacterial Toxins↗
PubMed1989

[Severe respiratory syncytial virus infections. Study of 87 infants hospitalized in an epidemic].

During a winter epidemic, 87 infants were admitted to Necker-Enfants-Malades hospital with a severe respiratory syncitial virus (RSV) infection. These infants fell into two groups: 37 infants without any medical history and 50 showing an underlying pathology (immune deficiencies, heart disease, CNS disorders, digestive malformations, allergic manifestations). Of the 37 infants with no medical history, most were below the age of 6 months and the RSV infection was manifested clinically by bronchiolitis or bronchitis. Most of the infants in the other group were more than 6 months of age and presented mostly with pneumonia or bronchiolitis. A respiratory distress syndrome was observed in 17 of the 87 infants, and virtually all of them were younger than 6 months. No significant difference was observed between the two groups with regards to the incidence of respiratory distress. Pulmonary infections complicating the course of the illness, most often due to commensal flora bacteria of the upper respiratory tract, were observed in 19 infants but with no greater frequency in the group at risk. Direct detection of viral antigens in nasopharyngal secretions not only enabled rapid diagnosis in all the infants but also allowed antiviral therapy to be started rapidly. Antiviral treatment by ribavirine, administered over a period of 5 days in 20 mg/ml doses by aerosol was instituted in 10 patients whose course might have become serious. In 8 of these patients, disappearance of the virus from secretions and recovery occurred. Two patients in the group at risk died despite treatment, with one case being considered a true therapeutical failure since the virus was still present in nasopharyngeal secretions.(ABSTRACT TRUNCATED AT 250 WORDS)

Child, Hospitalized↗
PubMed1988

Granulocyte-associated immunoglobulins in renal transplant recipients with cytomegalovirus infection.

Cytomegalovirus (CMV) infections in renal transplant recipients must be diagnosed rapidly, since they can be life-threatening unless chemotherapy is started early. Detection of granulocyte-associated immunoglobulins was compared with conventional virological methods for diagnosis of CMV infection in 71 renal transplant recipients. The granulocyte-associated immunoglobulin test (GAIT) was positive in 31 of 34 patients with proven CMV infections on the day of admission. By conventional virological criteria the diagnosis of active CMV infection could be made only 3-24 days later. The GAIT remained negative in 14 healthy transplant recipients, but it was positive in 9 of 23 patients with non-CMV-related post-transplantation complications. The GAIT, which is not a virological method, could be useful for rapid diagnosis of CMV infection; its sensitivity was 0.91 and specificity 0.82 (for patients without detectable immunoglobulins on erythrocytes or platelets) and the processing time is only 3 h.

Adult↗
PubMed1988

Invasiveness and intracellular growth of Listeria monocytogenes.

Listeria monocytogenes is an invasive bacterial pathogen capable of multiplying inside many host cells, including macrophages, enterocytes and hepatocytes. There is evidence to believe that secretion of listeriolysin O, an SH-activated exotoxin, is crucial for bacterial growth in host tissues. This exotoxin is stimulated in iron-deprived medium and mostly active at low pH (5.5). Electron microscopic studies showed that intracellular bacteria rapidly disrupt the vacuole membrane of phagosomes and freely multiply inside the cytosol of infected cells, thus escaping at an early stage of infection from the cellular microbicidal mechanisms. Vacuole disruption does not occur with a nonhemolytic mutant obtained by insertion of a single copy of transposon Tn1545 in the structural gene of listeriolysin O. These results strongly suggest that listeriolysin O is a major factor promoting intracellular growth of L. monocytogenes and that intracellular growth of virulent bacteria is initiated after escaping from the phagosomal compartment.

Animals↗
PubMed1987

T cell recognition of listeriolysin O is induced during infection with Listeria monocytogenes.

During bacterial multiplication, Listeria monocytogenes (strain EGD) secretes sulfhydryl-dependent cytotoxin, termed listeriolysin O, a virulence factor presumable promoting intracellular growth of this ubiquitous pathogen. The role of this exotoxin in the process of T cell activation was studied in vivo during the course of an experimental infection in the mouse. By using highly purified listeriolysin O, it was found that infection with viable, replicative bacteria induced in vivo the emergence of T cells specifically reacting against this exotoxin, as demonstrated by eliciting the expression of delayed-type hypersensitivity to listeriolysin O in Listeria-immune mice. The kinetics of this inflammatory reaction followed the same pattern as that observed with crude Listeria antigenic preparation classically used for the detection of delayed-type hypersensitivity, with a peak of expression by day 6 and a slow decline over the next 3 wk to a residual level, indicating the presence of memory T cells reacting with the exotoxin. This result, therefore, allowed us to identify for the first time that a pure immunogenic molecule secreted by L. monocytogenes is specifically recognized by sensitized T cells induced during the course of infection by L. monocytogenes. The expression of T cell-mediated immunity to listeriolysin O was generated by very low amounts of replicative bacteria, indicating that the exotoxin released in host tissues during the process of intracellular growth is highly immunogenic. Our data favor the view that the binding of listeriolysin O to the membrane cholesterol might be a critical event potentiating the in vivo expression of delayed sensitivity against this exotoxin. Indeed, the insertion of listeriolysin O into the cell membrane induced resistance to enzymatic proteolysis and membrane-bound listeriolysin O was significantly more effective in inducing delayed inflammatory reaction in Listeria-immune mice.

Animals↗
PubMed1987

Intracellular growth of Listeria monocytogenes as a prerequisite for in vivo induction of T cell-mediated immunity.

The in vivo induction of T cell-mediated immunity was studied by infecting mice with two genetically closely related mutants from Listeria monocytogenes, differing only with respect to the secretion of an active SH-dependent hemolysin. It is shown that even minute doses of hemolytic bacteria capable of growing in host tissues easily induced the expression of T cell-mediated immunity, as estimated by the level of delayed sensitivity, adoptive protection and long-lasting immunological memory. On the contrary, nonhemolytic bacteria unable to multiply in host tissues totally failed to initiate the expression of T cell-mediated immunity in vivo. This failure was even observed when mice were repeatedly infected by high doses of nonhemolytic bacteria, allowing to maintain a significant amount of viable bacteria for several days in host tissues. These results mean that the presence of viable bacteria at a significant level in the host is not sufficient per se to induce detectable T cell clonal expansion in the in vivo setting, implying that the process of bacterial growth inside macrophages is required to initiate in vivo the expression of T cell-mediated immunity.

Animals↗
PubMed1987

In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2.

Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.

Hemolysin Proteins↗