Peritoneal infection: a possible redox disease.

In a recent report, pertaining to Bacteroidesfragilis peritonitis, the influence of oxidation-reduction (redox) potential provided experimental evidence for B. fragilis penetration into Hela cell monolayers (using 3D imaging techniques). Bacteria grown under oxidizing conditions (+mV redox) penetrated into tissue cells unlike that of reducing conditions (-mV redox). The present results emphasise the significance of the level of redox potential during infection with an interpretation based on anaerobe/aerobe environmental flux, triggering the invasive mechanism.

Recruitment and derecruitment during acute respiratory failure: an experimental study.

We aimed to elucidate the relationships between pleural (Ppl), esophageal (Pes), and superimposed gravitational pressures in acute lung injury, and to understand the mechanisms of recruitment and derecruitment. In six dogs with oleic acid respiratory failure, we measured Pes and Ppl in the uppermost, middle, and most dependent lung regions. Each dog was studied at positive end-expiratory pressure (PEEP) of 5 and 15 cm H2O and three levels of tidal volume (VT; low, medium, and high). For each PEEP-VT combination, we obtained a computed tomographic (CT) scan at end-inspiration and end-expiration. The variations of Ppl and Pes pressures were correlated (r = 0.86 +/- 0.07, p < 0.0001), as was the vertical gradient of transpulmonary (PL) and superimposed pressure (r = 0.92, p < 0.0001). Recruitment proceeded continuously along the entire volume-pressure curve. Estimated threshold opening pressures were normally distributed (mode = 20 to 25 cm H2O). The amount of end-expiratory collapse at the same PEEP and PL was significantly lower when ventilation was performed at high VT. End-inspiratory and end-expiratory collapse were highly correlated (r = 0.86, p < 0.0001), suggesting that as more tissue is recruited at end-inspiration, more remains recruited at end-expiration. When superimposed pressure exceeded applied airway pressure (Paw), collapse significantly increased.

Relationship of Staphylococcal isolates in a Moroccan hospital by comparing phenotypical and genotypical tests.

The relationship of Staphylococcus isolates was determined among a collection of 26 clinical strains at the Centre Hospitalier Universitaire de Rabat. These isolates originated principally from blood culture and wounds. In order to affirm the clonal origins of these isolates, six phenotype (biotype, anti-biotype, serotype, phage type), and genotype (random amplified polymorphic DNA, pulsed field gel electrophoresis) methods were used. Biotyping, anti-biotyping, phage and serotyping were generally not sufficient while many isolates remained non-phage typeable. Random amplified polymorphic DNA analysis used in epidemiological typing seemed suitable for S. epidermidis and S. haemolyticus. However, rigorous standardization will be needed to assure reliable results. Pulsed field gel electrophoresis discriminated more efficiently than random amplification polymorphic DNA analysis. This study attests to the suitability of two or more methods in combination for typing Staphylococcus isolates.

Effects of hydrogen peroxide on growth and selected properties of Porphyromonas gingivalis.

In this study we first evaluated the effects of hydrogen peroxide (H2O2) on growth and selected properties of Porphyromonas gingivalis, and compared them with those obtained by a reducing agent (cysteine). The growth of P. gingivalis was only moderately affected when H2O2 was added at concentrations up to 30 mM in a complex culture medium. However, when a defined basal medium was used, H2O2 at a concentration of 3 mM completely inhibited growth of P. gingivalis. Incorporation of cysteine at concentrations up to 30 mM in both media had no effect on growth. The effects of H2O2 and cysteine on cell-associated hemagglutinating and Arg-gingipain activities were evaluated using bacteria grown in the complex culture medium. Both activities were strongly decreased when H2O2 was added in the assay mixtures. This inhibitory effect of H2O2 was reversible. On the other hand, including cysteine in the assay mixtures increased both activities. H2O2 and cysteine had no effect on the expression of heat shock protein (HSP)-68 and HSP-75 by P. gingivalis, as determined by SDS-PAGE and Western immunoblotting analysis. In the second part of the study, we tested whether growth of selected oral bacterial species may modify the oxidation-reduction potential (Eh) of the environment. It was found that certain species were able to either decrease (P. gingivalis, Fusobacterium nucleatum, Peptostreptococcus micros, Streptococcus mutans) or increase (Streptococcus sanguis) the Eh of the medium. Our study provides evidence that an oxidizing agent such as H2O2 may affect the biology of P. gingivalis. Moreover, growth of some members of the oral microflora can generate oxidizing and reducing conditions, and thus potentially influence the ecology of subgingival sites by affecting strictly anaerobic bacteria such as P. gingivalis.

Direct examination of subgingival plaque from a diseased periodontal site using confocal laser scanning microscopy (CLSM).

Material taken directly from a periodontal site was investigated using immunofluorescence, acridine orange staining and confocal laser scanning microscopy (CLSM). Porphyromonas gingivalis was tracked by a specific polyclonal antibody and its pronounced occurrence in inflamed as compared to non-inflamed areas was demonstrated. Further accompanying microorganisms were counterstained with acridine orange which could provide information on the viability of individual cells. Optical sections by laser microscopy revealed the spatial arrangement of the investigated material. The combination of specific staining and CLSM allows a detailed microbiological investigation of clinical material obtained directly without cultivation.

Biosynthesis of prostacyclin and thromboxane A2 during chronic hypoxaemia in children with cyanotic congenital heart disease.

The high risk of vaso-occlusive events in children younger than 4 years with cyanotic congenital heart disease and polycythaemia has been attributed to increased thromboxane (Tx) A2 formation. In older children with cyanotic congenital heart disease, however, the risk of vaso-occlusive events is much lower. We therefore hypothesized that the formation of TxA2 and prostacyclin is not disturbed in this age group. We measured urinary excretion of stable index metabolites of in vivo TxA2 and prostacyclin formation by gas chromatography-mass spectrometry in nine children (age 5.9-14.4, median 8.7 years) with cyanotic congenital heart disease, and in nine healthy, age-matched control subjects. The patients excreted less 2,3-dinor-TxB2 (systemic TxA2 formation, P = 0.03), 2,3-dinor-6-keto-PGF1 alpha (systemic prostacyclin formation. P = 0.03) and TxB2 (renal TxA2 formation, P = 0.01) than the control subjects. We conclude that in children older than 5 years with cyanotic congenital heart disease, endogenous synthesis of TxA2 and prostacyclin is not stimulated. This result may explain the lower risk of vaso-occlusive events in this age group as compared with younger children. In addition, our results suggest that chronic hypoxaemia may affect the in vivo formation of TxA2 and prostacyclin and the metabolic disposition of TxB2.

Lactate enhancement of sialylation of gonococcal lipopolysaccharide and of induction of serum resistance by CMP-NANA is not due to direct activation of the sialyltransferase: metabolic events are involved.

Lactate enhances lipopolysaccharide (LPS) sialylation and induction of serum resistance in gonococci by CMP-NANA. To investigate whether the enhancement is due to a direct effect on the sialyltransferase, an improved extraction of the enzyme and a reliable quantitative assay were devised. Gonococci (strain F62) were disrupted in a French pressure cell and the bacterial membranes were extracted for 1 h at 37 degrees C with a detergent, NONIDET (1% v/v). The assay involved sialylation of LPS by CMP-14CNANA and scintillation counting of the labelled LPS after fixing it on filter paper strips by trichloracetic acid (TCA) and washing away unincorporated CMP-14CNANA. It was rapid, reproducible and, although the enzyme preparations contained endogenous LPS, was dependent upon added LPS for maximum activity. At 37 degrees C the rate was constant for up to 5 min and proportional to the concentration of extract in the assay. A wide range of concentrations of lithium-L-lactate did not enhance the activity of the extracted sialyltransferase. At concentrations above 22 microM, it was inhibitory. Pre-incubation of gonococci with lactate enhanced subsequent LPS sialylation and induction of serum resistance by CMP-NANA. Hence, the process whereby lactate enhances the effect of CMP-NANA is separate from the action of CMP-NANA itself. Both processes were inhibited by a sublethal concentration of chloramphenicol, indicating that metabolic events are required. Evidently, the enhancement process does not involve a direct activation of the sialytransferase.

[Kinetics of sialylation of Neisseria gonorrhoeae by radiolabelled cytidine 5'monophospho-N-acetyl neuraminic acid]. / Cinétique de sialylation de Neisseria gonorrhoeae par l'acide cytidine 5'monophospho-n-acétyl neuraminique radio-marqué.

Neisseria gonorrhoeae (Strain A) induces a sialyl-transferase when treated with a very low concentration of cytidine 5'monophospho-N-acetyl neuraminic acid, 2 x 10(-3) nmol.ml-1, a concentration which is insufficient to produce an adequate resistance to human serum complement. The sialyl-transferase activity was detected by measurement of fixed 14C radio-labeled sialyl groups. Without this stimulation, there was practically no transfer of sialyl groups. The gonococcal sialyl-transferase could be considered inducible.

Kinetics of conversion of Neisseria gonorrhoeae to resistance to complement by cytidine 5'-monophospho-N-acetyl neuraminic acid.

Freshly isolated gonococci upon subculture are readily lysed by normal human serum although a few strains remain inherently resistant to the complement activity. The sensitive gonococci can be converted to serum resistance by incubation with a host derived factor referred to as cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA). These gonococci resist complement mediated killing due to their sialylation of an epitope structure on a component of lipo-oligosaccharide (LOS). In the present study, the kinetics of conversion to serum resistance by the action of sialyltransferase (STase) in Neisseria gonorrhoeae was followed with very low concentrations of CMP-NANA. This conversion could not be perceived at 2 x 10(-3) nmol.ml-1 but was fully attainable from 8 x 10(-3) to 2 x 10(-2) nmol.ml-1 CMP-NANA. When pretreated up to 100 min in presence of the very low concentration of 2 x 10(-3) nmol.ml-1, a potentiating effect on the conversion of gonococci by 2 x 10(-2) nmol.ml-1 was observed in relation to the time of preincubation. This action was abolished after exposure to a subinhibitory concentration of chloramphenicol (0.5 microgram.ml-1). The gonococci recovered their ability to convert to serum resistance following adequate washing. The potential for increase in STase activity should be of interest for understanding the conversion from a serum sensitive to a serum resistance state.

Effect of growth of Bacteroides fragilis at different redox levels on potential pathogenicity in a HeLa cell system: demonstration by confocal laser scanning microscopy.

During trauma, the intestinal anaerobe, Bacteroides fragilis, may enter into a pathogenic state. The process coincides with changing environmental conditions particularly the redox level in situ. To gain insight into this phenomenon B. fragilis was grown at different redox levels, and the invasive potential was examined using an in vitro model consisting of HeLa cell monolayers. The clinical strain AIP 5-86 was taken from a small collection of B. fragilis strains able to penetrate into tissue cell monolayers when selected by an acridine orange-crystal violet fluorescent staining technique. Following preliminary investigation by confocal laser scanning microscopy (CLSM), this particular strain was regarded as representative for examining the invasive potential. After growth in a defined medium under oxidizing, reducing or intermediate Eh7 conditions, the washed mid-log phase bacteria were allowed to interact with HeLa cell monolayers for 45 min at 37 degrees C. The results were extensively monitored by CLSM to follow the reactions in a stereoscopic dimension. In addition, the bacteria were examined by transmission and scanning electron microscopy before interaction to distinguish characteristics in surface configuration. The growth of the bacteria at particular redox levels seemed to influence their potential for pathogenicity. After growth at relatively high Eh, the bacteria easily penetrated into the HeLa cells, but not at low Eh, as determined by the laser scanning studies. Examination of the bacteria alone by transmission and scanning electron microscopy revealed small vesicles and a tendency to aggregate after growth at the low redox level while there were rather few vesicles and an implied dispersion at the high redox level. This leaves it open whether the invasiveness was based on the alterations found during growth of the bacteria. Different redox levels as well as the respective changes of the bacterial surface may help to discern the commensal from the pathogenic state of B. fragilis.

Demonstration by confocal laserscanning microscopy of invasive potential with Bacteroides fragilis.

The confocal laserscanning microscopy (CLSM) system provides a stereoscopic view of an object. By this system the penetration of B. fragilis into HeLa cells was observed. The intensity of contact is highlighted with time. The CLSM system consolidates the recently described fluorescence technique to test invasive potential. This work purports that certain gram-negative anaerobes should be considered for invasiveness.

The pH of gingival crevices and periodontal pockets in children, teenagers and adults.

Gingival crevice and periodontal pocket pH, measured directly with glass micro-electrodes, was near neutral at most sites in most individuals (mean pH 6.92 +/- 0.03 SEM, 69 subjects). Periodontal state ranged from healthy to periodontitis but neither clinical evidence of gingivitis at a site nor pocket depth were associated with crevicular pH different from that at healthy sites. This finding contradicts earlier reports that gingivitis is associated with a crevicular pH as alkaline as pH 9.06. Metallic antimony electrodes as used by earlier investigators were found to give pH readings that were too high by as much as 1.5 pH units in the presence of organic reducing agents of the type produced by oral bacteria within gingival crevices. In contrast, glass micro-electrodes respond only to hydrogen ions and thereby provided accurate measurements of pH even in the presence of organic reducing agents. Loss of CO2 to the atmosphere from biological fluids that are bicarbonate buffered resulted in a shift to alkaline pH by as much as 1 pH unit. As a result, only measurements taken within gingival crevices or periodontal pockets can provide accurate measurements of crevice or pocket pH.

A simple technique useful for clinical laboratory testing for invasive potential applied to Bacteroides fragilis.

A simple technique using fluorescent microscopy examines the association between Bacteroides fragilis and different types of tissue cells (epithelial, fibroblast, osteoblast). Through this stepwise intracellular staining and extracellular quenching some latent signs of invasive quality may be exhibited. The bacteria are incubated with monolayers of tissue cells, and the reactions immediately visualized after staining with acridine orange and masking by crystal violet. Intact bacterial rods exhibit a greenish hue which will reveal their location within the tissue cells but varies depending on tissue cell type. This direct means of testing for invasive potential applied to B. fragilis relies on the use of a readily available clinical tool.

Cytidine 5'-monophospho-N-acetyl neuraminic acid and a low molecular weight factor from human blood cells induce lipopolysaccharide alteration in gonococci when conferring resistance to killing by human serum.

Recently evidence has been obtained that a minute amount of cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) or a closely related compound is the low Mr factor in human red blood cells which induces Neisseria gonorrhoeae (BS4(agar] to resistance to killing by fresh human serum. Induction of gonococci to resistance by both CMP-NANA and semi-purified low Mr factor from red blood cells was accompanied by a 35-55% reduction of silver staining of lipopolysaccharide separated in SDS-PAGE gels of proteinase K digests. These alterations in lipopolysaccharide are probably responsible for conferring serum resistance. However, lipopolysaccharide-containing digests from resistant as well as from susceptible gonococci neutralised serum bactericidal activity. These observations may have wider implications since CMP-NANA is a sialylating agent wide-spread in mammalian tissues and LPS is ubiquitous amongst Gram-negative pathogens.

Hospitalization effect in acute mania.

We studied 31 patients meeting DSM-III criteria for bipolar affective disorder, manic type. All were treated in hospital, 18 on an open ward and 13 on a psychiatric intensive-care unit. Despite the use of only moderate doses of medication, dramatic clinical improvement was observed over the first 48 hours of treatment using the Brief Psychiatric Rating Scale and Beigel Mania Rating Scale as objective rating measures. Significantly greater improvement occurred in the psychiatric intensive-care unit in comparison to the open ward. We suggest that hospitalization effect is of prime importance in the early management of the acutely manic patient.