A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium.

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

Spread of Stenotrophomonas maltophilia colonization in a pediatric intensive care unit detected by monitoring tracheal bacterial carriage and molecular typing.

In our pediatric intensive care unit in Tours (France), intubated and ventilated inpatients are systematically monitored for tracheal bacterial colonization twice a week. This led us to detect five patients colonized with Stenotrophomonas maltophilia over a 4-month period. Molecular typing of the isolates using random amplified polymorphism DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) confirmed that four of the five isolates were genetically related. The strict isolation of carriers and improvements in hygiene measures stopped the spread. This systematic strategy prevented pulmonary nosocomial infections or allowed their early detection. Moreover, it has made it possible to assess the efficiency of care practices continuously.

Comparison of fecal flora following administration of two antibiotic protocols for suspected maternofetal infection.

Two protocols are used by French neonatologists for the treatment of suspected maternofetal infection (SMFI). Three groups of premature and term neonates were included to study the impact of antibiotics on fecal flora: 10 infants with SMFI treated with amoxicillin and netilmicin (group BI), 10 infants with SMFI treated with amoxicillin, cefotoxime and netilmicin (group TRI) and 10 infants without antibiotic therapy as controls (group C). Group BI samples were colonized with Klebsiella oxytoca and Escherichia coli resistant to amoxicillin and by Eneterococcus faecium and coagulase-negative staphylococci. In group TRI biodiversity of the intestinal flora was low, with rapid growth of staphyloccoci and occurrence of Candida spp. These modifications of the intestinal flora should encourage us to use antibiotic treatment as targeted as possible.

[Study of correlations between antibiotics prescriptions and guidelines in a neonatal intensive care unit]. / Etude de la corrélation entre les prescriptions d'antibiotiques et les recommandations dans une unité de soins intensifs néonatals.

UNLABELLED: The increasing use of wide spectrum antibiotics has been reported to be associated with a greater prevalence of multi-resistant bacteria. OBJECTIVES: The aims of this study were to survey the use of antibiotics and to evaluate the correlations between patterns of prescription of antibiotics and prescription guidelines in a neonatal intensive care unit. MATERIAL AND METHODS: In this 6-month study, all newborns admitted to the NICU and treated with antibiotics were included. Data regarding criteria of antibiotic prescription, length of treatment, and criteria of withdrawing treatment were collected. The correlation between prescriptions and guidelines was evaluated a posteriori by a non-prescriber physician. One hundred and sixteen newborns were included, of whom nine had received antibiotics on more than one occasion. Mean gestational age was 33.5 weeks. In 82% of cases, the reason for hospitalisation was respiratory distress syndrome. RESULTS: Patients received systemic antibiotics for primary infection (78%), nosocomial infection (17%) and postsurgical prophylaxis (5%). Suspected foeto-maternal infections (SFMI) were the dominant features of primary infection (96%). In 49% of cases, suspected infection was not proven and justified withdrawal of treatment within 3 days. Sixty percent of nosocomial infections occurred in newborns with gestational ages of less than 28 weeks. Bacterial criteria were decision-making factors only in nosocomial infections. An absence of observance of guidelines occurred in 9% of treated newborns, and in most cases involved excessive length of treatment. CONCLUSION: These results show: (1) the majority of antibiotic prescriptions were for not proven SMFI; (2) a low rate of nosocomial infections; (3) the predominance of nosocomial infections in premature newborns; (4) less than 10% of non-observance of guidelines. It appears necessary to develop more precise guidelines to limit antibiotic use and to evaluate them regularly.

[Staphylococcal pneumonia in a premature neonate: unusual transmission of nosocomial infection]. / Staphylococcie pulmonaire chez un prématuré : infection nosocomiale de transmission inhabituelle.

UNLABELLED: Late onset of neonatal infection could have been transmitted in prenatal period, but it is usually secondary to a postnatal transmission. CASE REPORT: A premature neonate developed staphylococcal pneumonia at 18 days of life. Genomic typing of the strains of Staphylococcus aureus obtained from the patient and from his mother (found in the endocervix culture 48 h before delivery) was identical. These strains were different from those isolated in other neonates colonised by S. aureus in the unit during at that moment. CONCLUSION: The observed case of staphylococcal pneumonia may correspond to a nosocomial infection secondary to a pre- or postnatal transmission of the agent by the mother.

Prevention of Candida colonization prevents infection in a neonatal unit.

This study represents a 1-year surveillance period using our epidemiology-based principles published and successfully followed since 1979: weekly culture for yeasts of oral and anal swabs, treatment with oral nystatin of all colonized newborns, and good hygiene/handwashing. Colonization was demonstrated in 23 out of 791 newborns admitted from October 1998 to September 1999. Twenty-two strains of Candida were identified: 16 C. albicans, 2 C. parapsilosis, 3 C. glabrata, and 1 C. tropicalis. Symptoms were erythema of the buttocks in 6 colonized newborns. No other culture positive for Candida could be found. Previous contamination was the main source (previous stay in an intensive care unit, rarely maternal origin). Contamination in the unit was unlikely. Eradication of Candida could be observed within 1 week. These good results, controversial in the literature, were obtained following epidemiological conclusions and support our guidelines.

[Necrotizing enterocolitis and apnoeas-bradycardias of the preterm newborn]. / Entérocolite ulcéro-nécrosante et apnées-bradycardies du nouveau-né prématuré

UNLABELLED: We conducted a case control study during six and a half years with the objective to analyse the risk factors for NEC. POPULATION AND METHODS: All cases of confirmed NEC matched to controls for identical gestational age and period of hospitalization; apnoeas-bradycardias prospectively counted. RESULTS: Forty-five cases were compared to 89 controls. The isolated risk factors were: an intra-uterine growth retardation (OR = 3,65, 95% confidence interval [CI] 95%: 1,54-8,63); a birth weight < 1000 g (OR = 8,16, CI 95%: 1,17-56,62), compared to a weight >/= 1500 g; a triple antibiotherapy (OR = 6,15, CI 95%: 1,16-32,45); an umbilical venous catheterization (OR = 2,64, CI 95%: 1,09-6,44); a number of simple apnoeas-bradycardias >/= 3rd tercile (n = 27) (OR = 4,54, CI 95%: 1,29-15,93), or severe (stimulated or with hypoxia) apnoeas-bradycardias >/= 3rd tercile (n = 8) (OR = 6,15, CI 95%: 1,59-23,75); an haemoglobin level lower than the 1(st) tercile (95 g/L) (OR = 5,90, CI 95%: 1,20-20,13); and milk thickening by Gumilk (OR = 2,78, CI 95%: 1,11-6,90). CONCLUSION: In the present practices, anoxo-ischemic factors during the first week of life do not represent an important risk of NEC; a great vigilance must be exercised for indications of the triple antibiotherapy and the treatment of apnoeas-bradycardias.

[Medical, cognitive and affective status of four-year-old very premature babies without severe disability. A case-control study]. / Devenir médical, cognitif et affectif à l'âge de quatre ans des prématurés indemnes de handicap sévère. Etude prospective cas-témoins.

AIM: To determine cognitive performances and affective development of a cohort of very premature babies at the age of 4 years. POPULATION AND METHODS: Longitudinal case-control study. Newborns less than 33 weeks gestation were included from 1992 to 1994 and were matched with two full-term newborns. At 4 years of age, they have been evaluated for medical status, cognitive functions with the K-ABC test and affective condition with the PSA and Conners' tests. RESULTS: The preterm group was significantly different from the controls for: a smaller height, (P < 0.01), a lower socioeconomic level (P = 0.027), a lower nursery school level (44% versus 27,6%, P = 0.05), the incapacity to draw "a bonhomme" (55,3% versus 93%, P < 0.001), lower performances for the K-ABC and PSA tests, a pathologic Conners score (20.4% versus 2.4%, P < 0.001). Multivariate analysis by logistic regression showed a similar neuropsychological results: a lower score of K-ABC (< 80, P = 0.0006, odds-ratio = 33.2), and a lower social competence score of PSA (< 45, P = 0.004, odds-ratio = 35.9). CONCLUSION: Follow-up of premature babies is required, and measures to prevent or minimize cognitive and social disabilities are mandatory.

Modification of gene expression induced in human osteogenic and osteosarcoma cells by culture on a biphasic calcium phosphate bone substitute.

Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-beta1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.

Incidence of Reye's syndrome in France: a hospital-based survey.

At the time of the study no information was available in France about the incidence of Reye's Syndrome (RS) and no warnings about RS and aspirin. The objective was to evaluate the incidence of RS in France by a hospital-based study. For a period of 1 year from November 1995 to November 1996, all French paediatric departments were required to report any child under 15 years with unexplained noninflammatory encephalopathy (i.e., CDC consciousness level stage I or deeper with normal CSF) and a threefold (or greater) increase in serum aminotransferase and/or ammonia. All suspected cases were classified by a panel of experts as probable RS or excluded RS. In 10% of randomly selected paediatric departments we checked that every suspected case had been reported. Forty-six suspected cases were reported during the year of the survey, of which 14 were classified as RS. Five of these 14 cases had a metabolic disorder. Nine children were definitively diagnosed as having RS (i.e., an estimated incidence of RS of 0.79/1,000,000 children, i.e., below 15/year). Eight children had been exposed to aspirin, four to aspirin alone and four to aspirin and acetaminophen. On the basis of these results the incidence of RS in France in 1996-1997 was not substantially different from that of countries where warning labels were already in use, but it was higher than in the US after 1994. This was probably due to the reduction in aspirin prescription in France because of warnings in Europe and the US and also because many cases of RS are now identified as metabolic disease. On the basis of these results and because the relationship between aspirin and RS has already been proved, public and professional warnings concerning RS on aspirin-containing products in cases of varicella and viral febrile illness have been adopted by the French Drugs Agency.

mRNA D(2) dopaminergic receptor expression after hypoxia-ischemia in rat immature brain.

Previous studies have shown a reduction of dopaminergic D(2) receptors (D(2)R) in the striatum after hypoxia-ischemia in newborn rats. We show here an early and transient reduction of mRNA D(2)R in nonatrophic brains following hypoxia-ischemia. The left carotid artery of P7 rats was ligated followed by hypoxia for 2 h. The rats were sacrificed after 24 h, 48 h and 14 days. D(2)R mRNA was studied by in situ hybridization, the cell number by conventional histology, and neuronal and astrocyte differentiation by immunohistochemistry. A 20% reduction of striatal mRNA D(2)R occurred 24 h after hypoxia-ischemia, whereas no reduction was observed after 48 h and 14 days. There were no differences in total cell number and in the expression of neuronal (MAP-1, MAP-2) and astrocyte (GFAP) markers between both brain hemispheres nor between control and hypoxia-ischemia animals. The early decrease in mRNA D(2)R could explain the delayed reduced D(2)R after neonatal hypoxia-ischemia.

Topical hyaluronidase decreases hyaluronic acid and CD44 in human skin and in reconstituted human epidermis: evidence that hyaluronidase can permeate the stratum corneum.

Hyaluronic acid (HA), a high molecular weight glycosaminoglycan of the extracellular matrix involved in growth, inflammation and wound healing, also contributes to the hydration and plastic properties of skin. Several drug and cosmetic formulations contain HA. We have initiated investigations that explore whether it is possible, by topical application, to modulate endogenous HA levels in skin. We developed a model epidermal culture system that exhibited a differentiated stratum corneum, and expressed HA and the HA receptor CD44, in a pattern similar to that observed in intact skin. Such in vitro skin equivalents are useful models for investigating the effect of topical drugs. HA and bacterial hyaluronidase were applied to the in vitro skin equivalent and to human skin. Their effects on endogenous HA and CD44 expression were examined using histochemical analysis. Topical HA treatment had no significant effect on HA or CD44 expression in either system. However, hyaluronidase decreased HA and CD44 expression in a dose-dependent manner in both the epidermal culture system and in skin. Apparently, HA is not able to permeate the epidermal culture system or human skin to a significant degree, but bacterial hyaluronidase does permeate both human skin and the culture system, depleting HA and decreasing CD44 expression. These effects were more prominent in the dermal than in the epidermal layers, suggesting that marked differences in HA metabolism exist in these two skin compartments. The ability of hyaluronidase to permeate the stratum corneum suggests that topical application may, additionally, be useful as a clinical modality.

Novel square planar copper(II) complexes with imino or nitronyl nitroxide radicals exhibiting large ferro- and antiferromagnetic interactions.

This paper reports the synthesis, crystal structures, and magnetic properties of two copper(II) complexes (1, 2) of general formula Cu(tfac)2(radical)2 (tfac = trifluoroacetate; radical = (1) 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NITPh) or (2) 2-phenyl-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazoline-1- oxyl (IMPh)). They crystallize in the monoclinic P2(1)/n space group with the following parameters: (1) a = 13.212(2) A, b = 9.136(1) A, c = 15.587(2) A, beta = 114.61(1) degrees, Z = 2; (2) a = 11.059(2) A, b = 15.289(1) A, c = 10.694(2) A, beta = 114.20(1) degrees, Z = 2. In both complexes the copper(II) ion is coordinated to two radicals in a slightly distorted square planar surrounding. The copper(II)-radical exchange couplings are antiferromagnetic for the nitronyl nitroxide (NITPh) complex (1) and ferromagnetic in the case of the imino nitroxide (IMPh) analogue (2). The ground state has been found to be a spin-doublet for 1 and the spin-quartet for 2. No thermal population of the highest states has been observed, indicating copper(II)-radical couplings of magnitude of J > 500 cm-1.

[Medical and psychological status of one-year-old premature babies without severe disability. Case-control prospective study]. / Devenir médical, psychologique et affectif à l'âge d'un an des prématurés indemnes de handicap sévère. Etude prospective cas-témoins.

BACKGROUND: This case-control prospective study was conducted to determine whether and how medical, psychological and affective development differs from premature to full-term newborns without severe disability. POPULATION AND METHODS: Newborns under or at 33 weeks gestation (W) were included from December 1992 to January 1994 and were matched with two controls. The same examiners evaluated each infant at the effective postnatal age of nine to ten months. RESULTS: Fifty premature babies (average gestational age [GA] = 30.7 W) were compared to 100 controls. The main problems were bronchopulmonary (P = 0.03) and sleep (P = 0.027) disorders. Motor disability was suspected in 9% of the cases and none control (P = 0.00003, OR = 3.44). By multivariate analysis, cases differed from the controls by infant-mother relation disturbances (OR = 13.3), motherhood anxiety (OR = 13.3), poor expressiveness (OR = 5.6), peripheral tonus anomalies (OR = 39.5) and sleep troubles (OR = 5.8). CONCLUSION: Premature newborns had risks for the child-mother relation but not for psychoaffective development disturbances.

Ca increase in secretory granules of stimulated mast cells.

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.

Consequences of pulmonary inflations (sighs) on cerebral haemodynamics in neonates ventilated by high-frequency oscillation.

High-frequency oscillation (HFO) is a technique frequently used in neonatal resuscitation, but which has yet to be evaluated. The use of intrathoracic pressures may have an effect on the cerebral circulation of immature neonates. The aim of this study was to examine the variations in cerebral blood velocity and oxygenation during brief pulmonary inflations (sighs), by focusing on alveolar recruitment. In this prospective study performed in 13 intubated and ventilated neonates (alpha = 5%; 1-beta = 80%), mean blood velocity and Doppler Resistance Index were measured, and variations in chromophores concentrations were evaluated by near infrared spectroscopy. Brief inflations at 4 cm H2O above the mean regulated intra-thoracic pressure did not cause any variation in the parameters measured. An explanation for this discordance with animal studies may be the level of pressure chosen, which could be more appropriate for the pulmonary compliance of neonates.