Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples.

Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.

Cyclic AMP and permeability coefficient of albumin of the isolated rat mesentery. Effects of Escherichia coli endotoxin.

We have investigated the mechanisms whereby Escherichia coli endotoxin exerts its exudative effects, by using an isolated rat mesentery placed as a separation membrane between the two compartments of a diffusion cell. The permeability coefficient of albumin (PA) can be easily computed from the equilibration rate of 125I-labeled albumin added to one compartment. E. coli endotoxin increased PA in a concentration-related manner. Direct measurements revealed an early and transient increase in cyclic AMP and prostaglandin E-immunoreactive material. These effects of endotoxin could be inhibited by indomethacin. Calcium-depleted tissues have a low PA, even though cyclic AMP levels could still be increased by endotoxin. It incubations were prolonged beyond 90 min, PA remained elevated, but prostaglandin E and cyclic AMP levels fell to control values. Similar results were observed with trypsin-treated tissues. These results suggest that transmesenteric passage of albumin is increased in the presence of endotoxin. During the earlier part of the incubation (up to 90 min), the effects could be related to a local synthesis of prostaglandin E, and are controlled by cyclic AMP and intracellular calcium levels. During longer incubations (90-280 min) mesothelial exfoliation could occur, allowing free diffusion of albumin through the remaining interstitial tissue.

The permeability coefficient of albumin of the isolated rat mesentery. Modification by some mediators of inflammation, cyclic AMP and calcium.

In order to study the mechanisms whereby mediators of inflammation exert their exudative effects, we used isolated rat mesentery placed as a separation membrane between the two compartments of a diffusion cell. In this experimental arrangement, the permeability coefficient of albumin (PA) can be easily computed from the equilibration rate of 125I-labelled albumin added to one compartment. Histamine, bradykinin, serotonin and prostaglandins A1, A2, E1, E2, F1 alpha and F2 alpha all increased PA to some extent, the maximal values being approx. +60%. Dibutyryl-cyclic AMP, theophylline and isoproterenol also increased PA, thus suggesting involvement of cyclic AMP. Direct measurements of this nucleotide confirmed this hypothesis; furthermore, a linear relation between cyclic AMP levels and PA could be demonstrated. In contrast, cyclic GMP is probably not involved in the control of PA. Calcium-depleted tissues had a low PA (approx. 40% below controls), and did not respond to exogenous prostaglandin E1. These results suggest that transmesenteric passage of albumin is at least partly controlled by cyclic AMP and intracellular Ca2+ levels.

Visible light C-H amidation of heteroarenes with benzoyl azides.

Benzoyl azides were used for the direct and atom economic C-H amidation of electron rich heteroarenes in the presence of phosphoric acid, a photocatalyst and visible light. Hetero-aromatic amides are obtained in good yields at very mild reaction conditions with dinitrogen as the only by-product. The reaction allows the use of aryl-, heteroaryl- or alkenyl acyl azides and has a wide scope for heteroarenes, including pyrroles, indole, furan, benzofuran and thiophene giving good regio-selectivities and yields.

Controlled fall in natremia and risk of seizures in hypertonic dehydration.

The aim of the study was to derive some practical measurements which might help in defining a "safe" infusion rate in order to avoid seizures during treatment of hypernatremic dehydration. Forty seven infants with hypernatremic dehydration were rehydrated on a 160 ml/kg/24 h basis: 9 developed seizures during treatment (group I), 22 matched for age did not convulse (group II). Nine subsequent cases were prescribed a 120 ml/kg/24 h regimen: none convulsed (group III). The three groups were comparable in many respects, including initial plasma Na and pH. Fluids were comparable regarding (Na), their rates of administration were respectively 216, 181 and 123 ml/kg/24 h. The rate of infusion affected slopes of decreases in natremia. It was suggested that the decrease in plasma Na should not exceed 0,5 mEq/1/h.

Thermal behavior and elastic properties of phospholipid bilayers under the effect of a synthetic flavonoid derivative, LEW-10.

We have investigated the effect on phospholipidic bilayers of LEW-10, a synthetic flavonoid, derivative of diosmin. Two optical techniques, Quasi-elastic Light Scattering (QLS) and Fourier Transform Infrared Spectroscopy (FT-IR) were used. The results show that in the presence of LEW-10, the phase transition of the bilayers is lowered and that the elastic modulus is decreased. The FT-IR results indicate interactions in the aqueous interface regions of the bilayers. We also discuss LEW-10 comparatively with another derivative, LEW-7/S1, whose effect has been previously studied.

Randomised, controlled trial of concomitant pneumococcal and meningococcal conjugate vaccines.

A randomised, open-label study compared the immunogenicity and safety of 7-valent pneumococcal conjugate vaccine (PCV7) and meningococcal C conjugate vaccine (MnCC vaccine) administered concomitantly and individually. Infants received PCV7+MnCC vaccine (n=265), PCV7 alone (n=268) or MnCC vaccine alone (n=178). PCV7 was administered at 2, 3½, 6 and 12 months, and MnCC vaccine at 2, 6 and 12 months. For the 7 pneumococcal serotypes tested (4, 6B, 9V, 14, 18C, 19F and 23F), proportions of subjects with pneumococcal serotype-specific immunoglobulin G (IgG) antibody concentrations ≥0.35 µg/mL post-infant series were non-inferior for the PCV7+MnCC vaccine (91.5-99.6%) and PCV7 (89.0-99.6%) groups. Proportions of subjects achieving serogroup C meningococcal serum bactericidal assay titres ≥1:8 post-infant series were non-inferior for the PCV7+MnCC vaccine (99.6%) and MnCC vaccine groups (98.8%). Pneumococcal IgG antibody levels were similar in the PCV7+MnCC vaccine and PCV7 groups at each time point. Post-infant and post-toddler meningococcus C serum bactericidal assay titres and IgG levels were similar in the PCV7+MnCC vaccine and MnCC groups, although pre-toddler, the levels were lower in the PCV7+MnCC vaccine group than the MnCC vaccine group. Immune response rates to diphtheria antigen approached 100% for all vaccine groups. Local reactions were mostly similar among the treatment groups. The MnCC vaccine group had lower rates of some systemic events than the PCV7+MnCC vaccine group. Immune responses to PCV7+MnCC vaccine were non-inferior compared with those seen with each vaccine administered alone.

[Pentose phosphate pathway and nucleic acid synthesis in human funicular tissue. In vitro studies]. / La voie des pentoses et la synthèse des acides nucléiques dans le tissu funiculaire humain. Etudes in vitro

Umbilical cord slices were incubated with either 1- or 6-14C-glucose, the radioactivities of which were measured in CO2 evolved. The ratio, 1 CO2/6 CO2 was low, being comprised between 1 and 2. Furthermore, this incubation of cord slices with tritiated uridine or thymidine resulted in very low incorporations, especially for the latter. Therefore, both the pentose phosphate pathway and the synthesis of nucleic acid have a low activity in the cord tissue: these might be signs of senescence in this otherwise fetal organ.

[Oxidative metabolism of funicular tissue. I. Effect of exogenous substrates on oxygen consumption of funicular tissue in vitro]. / Le métabolisme oxydatif du tissu funiculaire. I. Effets de certains substrats exogènes sur la consommation d'oxygène du tissu funiculaire in vitro

Umbilical cord slices were incubated with various concentrations of either glucose, pyruvate or succinate. Furthermore the conversion of pyruvate to AcCoA and the oxidation of the latter were assessed respectively by measuring labelled CO2 evolved from 14C-pyruvate labelled on C1 or C2. The results suggest that 1) ADP stores are low since glucose induces a Crabtree effect; 2) the "succinoxidase" complex is far from being saturated and cannot account for the low oxidations observed under basal conditions; and 3) that the limiting step should be looked for either in Krebs' cycle or at the level of Complex I of the electron transport chain.

[Oxidative metabolism of funicular tissue. II. Determination of the activity of certain Krebs cycle enzymes]. / Le métabolisme oxydatif du tissu funiculaire. II. Détermination des activités de certaines enzymes du cycle de Krebs

Previous experiments have suggested that a partial metabolic block might restrain the oxidative metabolism of the cord tissue between the decarboxylation of pyruvate and the oxidation of succinate. Some of the dehydrogenases of the Kreb's cycle were assayed on acetone powders prepared from human cords. Isocitrate dehydrogenases (both NAD and NADP-specific) have much lower activities than the alpha-ketoglutarate- and malate dehydrogenases; a partial block might be located at this level. Moreover, the malic enzyme has a rather high activity, and might play a significant role by regenerating NADPH in a tissue where other sources of this coenzyme are practically absent.

The rabbit during the last third of gestation. Data concerning the whole fetus, its diaphragm and brown adipose tissue.

Total lipid, protein and glycogen concentrations were determined for the diaphragm and interscapular brown adipose tissue of fetal (22-31 days of gestation) and neonatal rabbits. In the diaphragm, protein concentration doubles and lipid concentration drops to half its initial value during the period under study; the glycogen level reaches its maximal value at 27 days, and decreases thereafter. In the brown fat, the lipid concentration rises steadily until the immediate prenatal period; the protein concentration remains steady and the glycogen is highest at 29 days of gestation. Fetal plasma glucose and insulin levels increase steadily; there exists a positive correlation between fetal plasma insulin levels and fetal body weight.

Involvement of prostaglandins in the local action of endotoxin.

Endotoxin from Gram-negative bacteria increases the permeability coefficient of albumin in isolated rat mesenteries used as a separating membrane between the two halves of a diffusion cell. Endotoxin also promotes cyclic AMP accumulation in similar mesenteric sheets. These effects are dose-related, and are inhibited by indomethacin. As shown by direct assay, Prostaglandin E-immunoreactive material is synthesized in the presence of endotoxin and is responsible for the increase in albumin permeability and for the increase in cyclic AMP.

[Increased vascular permeability during endotoxinemia in the child (author's transl)]. / Accroissement de la perméabilité vasculaire lors de l'endotoxinémie chez l'enfant.

There is no uniform agreement about the effects of bacterial endotoxins upon vascular permeability. We presently report some clinical observations indicating an increase in vascular permeability during sepsis and shock related to Neisseria meningitidis in children. 133 children, admitted for a severe infection due to Neisseria m. were separated into three groups according to the severity of the initial clinical picture. The patients presenting with a sepsis or shock had significantly lower mean plasma protein levels on admission. This was not related to any hemodilution or caloric deficiency. Hypoproteinemia worsened during the 24 first hours of treatment, despite hemoconcentration as presented by some patients [12]. The speed of decrease in protein concentrations was inversely related to the molecular weights of the different protein fractions. These observations indicate an increase in vascular permeability with maintainance of vascular membrane selectivity to macromolecules during early meningococcic septicemia and shock.

The role of various prostaglandins on the correlation between permeability to albumin and cAMP levels in the isolated mesentery.

Prostaglandins (PG) have been shown to raise the level of cyclic AMP (cAMP) in various tissues, and to increase permeability. Whether both events are linked, is at present a matter of speculation. We have investigated the effects of PGE1, E2, A1, A2, F1alpha and F2alpha on an isolated rat mesentery placed in a diffusion cell (surface area : 2 sq.cm). The PGs (5 microgram/ml) increased the passage of (I 125) - Albumin across the mesentery. In other experiments, diks of rat mesentery (surface area : 2 sq.cm) have been incubated in assay tubes, and cAMP levels measured by a binding protein assay. We have observed an excellent correlation between increases in permeability and cAMP levels (r=0.961). In order of increasing potency on both parameters, the PGs may be classified as follows : PGF, PGA and PGE. In the rat mesentery, under the influence of prostaglandins, increases in permeability and in cAMP levels are apparently connected.

[Correlation between permeabiltiy to albumin and levels of cyclic adenosine monophosphate in the incubated rat mesentery]. / Corrélation entre la perméabilité à l'albumine et les taux d'adénosine monophosphate cyclique dans le mésentère incubé du rat

Histamine, bradykinin and prostaglandin E1 increase the permeability coefficient of albumin; this effect is mimicked by dibutyryl-cAMP and by theophylline. They also cause a significant rise in the cAMP levels of the incubated tissue. Serotonin has lesser activities on these parameters. Presumably, the first three mediators control the transcellular passage of albumin by increasing the cAMP content of the tissue, serotonin being less effective. These findings on the mesentery seem to have a couterpart in vascular tissue as shown by others using different methods.

[Mechanisms of acute pulmonary edema during meningococcal shock in children (author's transl)]. / Mécanismes d'apparition de l'oedème pulmonaire aigu lors du choc méningococcique de l'enfant.

Meningococcic shocks may induce fatal acute pulmonary edemas. Ten children, who died in this condition, have been compared to 19 others, who during the treatment of the shock, developed right heart failure, without acute pulmonary edema (8 deaths, 11 survivals). The three groups of children could not be differentiated regarding their clinical status on admission, during the shock phase, or the treatments administered. Composition, volume and speed of administration of fluids were similar in the three groups. An increase in pulmonary capillary permeability could have occurred, and lead to the development of acute pulmonary failure, as presented by the children of the first group.

[Mechanisms of acute pulmonary edema during meningococcal shock in children (author;s transl)]. / Mécanismes d'apparition de l'oedème pulmonaire aigu lors du choc méningococcique de l'enfant.

Meningococcic shocks may induce fatal acute pulmonary edemas. Ten children, who died in this condition, have been compared to 19 others, who during the treatment of the shock, developed right heart failure, without acute pulmonary edema (8 deaths, 11 survivals). The three groups of children could not be differentiated regarding their clinical status on admission, during the shock phase, or the treatments administered. Composition, volume and speed of administration of fluids were similar in the three groups. An increase in pulmonary capillary permeability could have occurred, and lead to the development of acute pulmonary failure, as presented by the children of the first group.

Incorporation of amino acids into protein of foetal rabbit diaphragm: effect of insulin.

The incorporation of six L-amino acids into the protein fraction of the diaphragm of foetal and neonatal rabbits has been measured. Insulin stimulates the incorporation of lysine from the 29th day and of glycine from the 31st day of gestation onwards. The incorporation of histidine, serine and threonine is only enhanced in the diaphragm of 16-20 hour-old rabbits; no effect of insulin on the incorporation of leucine has been shown for the period under study.