[Anemia, a new severity factor in young infants with acute viral bronchiolitis?] / L'anémie, un nouveau facteur de sévérité de la bronchiolite aiguë du nourrisson ?

INTRODUCTION: The role of anemia is raised as a risk of low respiratory infection of the child, but there are no data on anemia as a severity factor in acute viral bronchiolitis (AVB) in infants. METHODS: All infants less than 16 weeks old admitted to Montpellier University Hospital from 2015/10/01 to 2016/04/01 for AVB were included in a retrospective observational study. The primary objective was to determine whether the hemoglobin (Hb) concentration on admission was an independent factor of clinical severity, judged by the modified Wood's clinical asthma score (m-WCAS). The secondary objective was to assess the impact of Hb level on the characteristics of hospitalization, including the type and duration of respiratory support. RESULTS: The m-WCAS was used at least once during hospitalization in 180 out of 220 patients (82%), making it possible to distinguish patients with mild AVB (maximum m-WCAS<2, n=81) from patients with severe AVB (maximum m-WCAS>2, n=99). A logistic regression model indicated that the Hb concentration, for every 1g/dL decrement, was an independent factor of AVB severity (OR 1.16 [1.03-1.29], P=0.026). A level under 10g/dL on admission was associated with a higher use of continuous positive airway pressure (P<0.001), as well as a longer duration of respiratory support (P=0.01). CONCLUSION: This study suggested that anemia may influence the clinical expression of AVB in young infants.

Macroparasites of Pallas's squirrels (Callosciurus erythraeus) introduced into Europe.

Introduced pets released in natura can lead to sanitary risks for native fauna and humans. We analysed the macroparasite fauna of a total of 49 Pallas's squirrels, Callosciurus erythraeus, from two populations introduced into urbanised areas in Europe (n=16 female symbol and 13 male symbol from Antibes, France, 43 degrees 33'N-7 degrees 7'E; n=11 female symbol and 9 male symbol in from Dadizele, Belgium, 50 degrees 52'N-3 degrees 5'E). Of the 185 identified ectoparasites from Antibes, 183 were sucking lice Enderleinellus kumadai, with male squirrels 10 times more intensely infested than females. The flea Nosopsyllus fasciatus was found on two hosts. No hard ticks were recovered. Of the 131 arthropods specimens from Dadizele, 45 belonged to E. kumadai, with male squirrels three times more intensely infested than females. Eighty-six arthropods belonged to another sucking louse, Hoplopleura erismata, with males infested twice as intensely as females. No fleas or hard ticks were found. We only found 12 immature Hymenolepis sp. cestodes in the small intestine of three squirrels from Antibes and two immature Mastophorus sp. female nematodes in the stomach of a squirrel from Dadizele. We found no other helminths in the body cavity, heart, lung, liver, kidney or bladder. The macroparasite fauna of these two squirrel populations is consistent with what is expected from an introduced host, i.e., a few species dominated by specialist taxa imported with founders. The scarcity of other rodent species in the urbanized areas where Pallas's squirrels were sampled may explain the low variety of newly acquired macroparasites. The discrepancy in sucking lice infestations between males and females could be due to differences in either behaviour or physiology in this non-sexually dimorphic host. Based on the macroparasites found in this study, we expect minimal sanitary risks for both native fauna and humans in urbanized habitats such as those in our study.

Does phosphoryl protonation occurs in aqueous phosphoesters solutions.

Ionisation of trimethylphosphate (TMP), dimethylphosphate (DMP) and diethylphosphate (DEP) is investigated by acidic titration in water by Raman (R), Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies. The vibrational frequencies of the PO(2)(-) ionic form and the neutral form were found in accord with the literature. While increasing further H(+) concentration, the PO band disappears in the benefit of new ones. These results, together with deuteration experiments indicate the presence of a new ionic form positively charged with general formula R(1)R(2)R(3)P(OH)(+) or R(1)R(2)P(OH)(+)(2). The pK of this phosphonium entities is lying in the range -2, -4. These results were confirmed by (31)P NMR titration. The occurrence of such a phosphonium ion in aqueous solutions might be of crucial importance for biochemical reactions and interactions, owing to the large spread of phosphoryl group in biomolecules and keeping in mind that intracellular compartments are more likely concentrated media with little free water than real aqueous solutions. Furthermore, pK's can be shifted by physical-chemical parameters like dielectric constant and electric field. This may involve at least fractional positive charge apparition that might be important in biochemical regulation by charge-charge and charge-dipole interactions. This finding will gain to be further explored on more complex molecules like phospholipids, nucleic acids and proteins.

Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.

Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.

Enzyme immunoassay of staphylococcal enterotoxins in dairy products with cleanup and concentration by immunoaffinity column.

Two different immunoaffinity columns (IACs) were prepared for detection of staphylococcal enterotoxins (SETs) from dairy products. First, a specific IAC for staphylococcal enterotoxin A (SEA), IAC-1, was prepared by coupling monoclonal antibody (mAb) directed against SEA; second, a polyspecific IAC for SEA, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SECs), and staphylococcal enterotoxin D (SED), IAC-2, was prepared by coupling a mixture of mAbs against SEA, SECs, and SED, and rabbit IgG against SEB. These columns were applied for detection of SETs in dairy products, after extraction, immunoaffinity chromatography, and enzyme immunosorbent assay (EIA). Overall recoveries from dairy products spiked with 1 ng SEA/25 g averaged 81.2% (range, 76-85%) on IAC-1. The repeated use of IAC-1 was then determined with good efficiency of 91.5%, in more than 10 runs. On the other hand, a recovery yield of 77% of SETs (SEA, SEB, SEC, and SED) from dairy products spiked with 2.5 ng of each enterotoxin per 25 g, was obtained with IAC-2. IAC-2 was also successfully subjected to the chromatography of naturally contaminated foods implicated in staphylococcal food poisoning outbreaks. This new extraction-concentration-immunoaffinity-chromatography method (ECIC) is very useful for improving staphylococcal enterotoxin detection and eliminating matrix effect in EIA of dairy products.

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

Behaviour and enterotoxin production by Staphylococcus aureus during the manufacture and ripening of raw goats' milk lactic cheeses.

To study the possible presence of staphylococcal enterotoxin A in raw goats' milk lactic cheese, milk was inoculated with an enterotoxigenic Staphylococcus aureus strain to a final concentration of 4, 5 and 6 log(cfu/ml). Cheese was prepared following industrial specifications and ripened for 42 d. Detection of the enterotoxins was by the Vidas Staph enterotoxin test (BioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. Staphylococcal counts declined markedly after draining, and by the end of ripening they had disappeared from some cheeses. In contrast, aerobic mesophilic organisms grew well. The level of staphylococcal enterotoxin A recovered varied from 1 to 2.5 ng/g cheese made with an initial population of 10(5) or 10(6) cfu/ml. Only traces of enterotoxin A (0.5 ng/g) were detected in cheeses made with the lowest Staph. aureus inoculum used in this study. Enterotoxin A was also detected in cheeses from which Staph. aureus had disappeared.

Diarrhoeal toxin production at low temperature by selected strains of Bacillus cereus.

The growth of four Bacillus cereus strains producing diarrhoeal toxin at 32 degrees C (F4433/73 and 29.155, isolated on the occasion of foodborne outbreaks, and F4581/76L and F4581/76R, two variants of a clinical strain), a weakly toxigenic strain isolated in routine analysis of food (3505M) and an emetic isolate (F3502/73) was investigated at low temperature. Biomass was determined by protein assay. Generation times were: for strain F3502/73, which grew at > or = 12 degrees C, 8.71 h (at 12 degrees C); for other strains, which grew at > or = 10 degrees C, 10.2 to approximately 18.9 h (at 10 degrees C). Toxin production during growth was evaluated by a commercial kit (Oxoid) and by a toxicity test on Chinese hamster ovary cells. Strains F4433/73 and F4581/76, secreting high levels of diarrhoeal toxin during the exponential phase at 32 degrees C, produced high levels of toxicity at 10 degrees C until the stationary phase. Strain 29.155 had decreased toxin production at 10 degrees C. Toxicities for cellular extracts remained low when compared with culture filtrates. A correlation was found between the toxicity values given by the two detection methods tested, and the suitability of both methods for the detection of potential poisoning isolates is discussed.

Immunoenzymatic detection of staphylococcal enterotoxins: international interlaboratory study.

An international interlaboratory study was performed by 13 laboratories to validate a commercially available, rapid enzyme immunoassay for detection of staphylococcal enterotoxins A, B, C, D, and E in foods. The 5 enterotoxin serotypes were detected at a level of 0.5 ng/g in mushrooms and ravioli, 0.8 ng/g in meat, 1 ng/mL in milk, and 1.5 ng/g in raw milk cheese when these foods were artificially contaminated with enterotoxin A. Enterotoxins A, B, C, D, or E were also detected in culture supernatants with no protein A interference when normal rabbit serum was used. This method was validated by the French Normalization Agency for the identification of staphylococcal enterotoxins in foods and culture fluids.

Enterotoxin production by coagulase-negative staphylococci isolated from goats' milk and cheese.

An antigen related to the Enterotoxin E from Staphylococcus aureus was produced by ten of 187 coagulase-negative staphylococci (CNS) isolated from goats' milk, whey and cheese in quantities ranging from 10 to 90 ng/ml supernatant. The enterotoxin-producing strains were identified at the species level as S. simulans, S. xylosus, S. equorum, S. lentus and S. capitis. Detection of the enterotoxins was done by the VIDAS SET test (bioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. The results obtained were further confirmed by Southern blotting, using two radioactive oligonucleotide probes that hybridized specifically with the gene of S. aureus coding for the enterotoxin E.

Production of diarrheal toxin by selected strains of Bacillus cereus.

The production of diarrheal toxin by six selected strains of Bacillus cereus was monitored during growth at 32 degrees C, a temperature described as near-optimal for growth and toxin production. Toxic activity was measured in culture filtrates and cellular extracts sampled at three different times during growth. Two alternative methods, a cytotoxicity test on Chinese hamster ovary (CHO) cells and a commercial immunological test (BCET-RPLA, Oxoid) were used. Toxin titres were in agreement with epidemiological characteristics and toxicity demonstrated by using other systems in other examinations. A comparison of intra- and extracellular toxicities measured at the exponential and stationary growth phases showed that the toxin was essentially secreted during the exponential phase. For several strains, secretion peaked during the period from the middle exponential phase to the beginning of the stationary phase. There was no important overall increase of the toxicity during full and late stationary phase. The level was stable or even lower, thus indicating that diarrheal toxin production during stationary phase was small, if any, and that the toxin was unstable under these conditions. Statistical analysis of toxicities showed that the cytotoxicity test was correlated with the immunological test (significant at a 1% level). For routine determinations, a toxicologic laboratory may use any of the two methods, depending on its facilities, the immunological test being relatively expensive.

Intravenous immunoglobulin (IVIg) modulates the expansion of V beta 3+ and V beta 17+ T cells induced by staphylococcal enterotoxin B superantigen in vitro.

The authors investigated the effect of IVIg on T-cell proliferation induced by the superantigen, staphylococcal enterotoxin B (SEB). The addition of IVIg to normal PBMC stimulated with SEB resulted in a threefold increase in the proportion of CD3+ blast cells expressing V beta 3 and V beta 17, two subsets of T cells that selectively expand in the presence of SEB. There was no increase in the proportion of T-cell blasts expressing V beta 2, V beta 8 and V beta 13.6 antigens that do not respond to SEB in the absence of IVIg. As described previously, IVIg inhibited T-cell proliferation independently of V beta specificity. The effects of IVIg were mediated by variable regions of immunoglobulins since they were reproduced with F(ab')2 fragments of IVIg but not with purified Fc fragments of IgG. The observation that SEB-activated T cells are rescued from inhibition of proliferation by IVIg indicates that Mg modulates the effects of superantigen on T cells. These results may be of relevance for understanding the mechanisms underlying the effects of IVIg in patients with diseases in which T-cell superantigens are of pathophysiological significance.

Activity of rifabutin, clarithromycin, ethambutol, sparfloxacin and amikacin, alone and in combination, against Mycobacterium avium complex in human macrophages.

Disseminated infection with Microbacterium avium complex (MAC) in patients with AIDS is currently treated with a combination of antimycobacterial agents in order to prevent the selection of resistant mutant strains. Although clinical and microbiological responses can generally be achieved within a few weeks, relapses are common and require modification of the combination regimen or identification of effective alternate therapies. In this study we investigated the activities of rifabutin 0.5 mg/L, sparfloxacin 1 mg/L, clarithromycin 4 mg/L, amikacin 16 mg/L and ethambutol 2 mg/L, alone and in combination, against nine strains of M. avium isolated from the blood of patients with AIDS in order to identify regimens with the greatest therapeutic potential. Macrophages derived from human monocytes were infected with M. avium and inoculated with a single drug or a combination of drugs; cfu counts were performed at 0, 4 and 7 days after infection. At day 4 and at day 7, the combination of rifabutin, clarithromycin, amikacin and sparfloxacin displayed the highest degree of activity. However, the activity did not differ significantly from that of the combination of rifabutin, clarithromycin and ethambutol. The results of this study confirm the activity of combinations including rifabutin and clarithromycin (+/- ethambutol) in human monocyte-derived macrophages and suggest potentially useful associations in incorporating sparfloxacin and amikacin.

Pharmacokinetic study of fentiazac and its main metabolite hydroxyfentiazac in the elderly.

The pharmacokinetics of fentiazac (F, CAS 18046-21-4) and hydroxyfentiazac (OH-F) were estimated in 12 elderly (> 76 years). After an oral single dose of 200 mg F, the plasma and urine profiles were determined using high-performance liquid chromatography with a fluorescence detection. When compared to results obtained in young adults, the maximum plasma concentrations (5.4 +/- 1.9 mg/l) and-the time to reach them were identical. The terminal half-life (7.0 +/- 9.1 h) was longer, due to a slight increase of the apparent volume of distribution and a decrease of the elimination clearance. The findings suggest that the dosage regimen of this drug should be decreased in the elderly. Moreover, the variability of the pharmacokinetics being larger, individual adaptation of the daily dose should be performed.

Production and characterization of monoclonal antibodies to staphylococcal enterotoxins: use in immunodetection and immunopurification.

Four cell lines producing monoclonal antibodies were obtained by fusion of NS1 myeloma cells with splenocytes of BALB/C mice immunized with only 1 microgram of each staphylococcal enterotoxin A, B, C1 and D by a modified technique of intrasplenic boosting. This procedure was considerably more efficient than the more commonly used intravenous boosting. The antibodies EC-A1, EC-B1, EC-C1 and EC-D1, all of the IgG1 subclass, have high affinities for the corresponding enterotoxins A, B, C1 and D, with dissociation constants of 1.4, 2.8, 1.4 and 1.5 nM respectively; in addition EC-B1 showed a high affinity (2.1 nM) for enterotoxin C1. All these antibodies recognize, by immunoblotting, the homologous purified enterotoxins as well as enterotoxins from the bacterial culture supernatants. A rapid indirect double sandwich ELISA using a pair of antibody preparations was developed, where monospecific monoclonal antibodies were used to coat plastic plates and polyspecific rabbit antibodies were used to detect the enterotoxins under field conditions. These antibodies which are capable of immunoadsorbing the enterotoxins from staphylococcal culture filtrates and from natural fluids such as milk, were used to immunopurify enterotoxins A, C1 and D. The homogeneity and integrity of the affinity purified toxins A, C1 and D was verified by direct automated Edman degradation and yielded single amino terminal sequences which were moderately homologous to those published previously for B and C1 enterotoxins.