Initial Specimen Diversion Device® reduces blood culture contamination and vancomycin use in academic medical centre.

BACKGROUND: In suspected bloodstream infections, accurate blood culture results are critical to timely diagnoses and appropriate antibiotic administration. AIM: An Initial Specimen Diversion Device®, Steripath® (Magnolia Medical Technologies, Seattle, WA, USA) was evaluated for efficacy in reducing blood culture contamination at Brooke Army Medical Center (6.8% six-month contamination rate prior to intervention) in a six-month quality improvement project. METHODS: Blood cultures in the emergency department were collected using either Steripath or the standard method. Blood samples of 20 mL were cultured into an aerobic and anaerobic medium and incubated for five days using an automated microbial detection system immediately after collection. Positive bottles were Gram-stained and plated. Rapid molecular polymerase chain reaction identification was performed on all first positive bottles within a blood culture set for each admission or ED visit. Speciation was deduced during antimicrobial sensitivity testing using the Vitek-2 instrument. FINDINGS: Seven out of 1016 (0.69%) contamination events occurred when using Steripath vs 53 out of 800 (6.6%) contamination events when using the standard method. Steripath use was associated with a 90% lower incidence of blood culture contamination vs the standard method. Post study, Steripath use was implemented as standard practice hospital-wide, and a retrospective data analysis attributed a 31.4% decrease in vancomycin days of therapy to Steripath adoption. CONCLUSION: Using Steripath significantly decreased blood culture contamination events for bacterial bloodstream infections compared to the standard method. Subsequent adoption of Steripath reduced overall vancomycin usage. With widescale implementation Steripath could bolster antibiotic stewardship, mitigating antibiotic resistance caused by unnecessary antibacterial treatments.

Residential crowding and asthma in preschool children, a cross-sectional study

Background: Asthma is the most common chronic disease in children, and associations with crowding have been reported. The aim of this study was to explore possible associations of crowding with asthma in children. Methods: Seven cross-sectional surveys with preschool children were conducted within the framework of the health monitoring units in Bavaria, Germany, from 2004 to 2014. Residential crowding was defined as habitation of more than one person per room or less than 20m2 living space per person. Logistic regression models examined temporal changes in crowding, applying the first survey as reference. The relationship between crowding and physician-diagnosed asthma, asthma defined by the International Study of Asthma and Allergies in Childhood (ISAAC) and asthma symptoms were analyzed. Results: Analyzing temporal changes of crowding rates did not reveal any differences over the years. However, the stratified descriptive analysis indicated a crowding increase in time in urban households where parents had a low education level (47.9% in 2004/05, 55.8% in 2014/15). No association was found between crowding and the variables "physician-diagnosed asthma" in 2014/15, "asthma defined by ISAAC" in 2014/15, or "wheezing" in 2014/15. A positive association with cough was identified in 2014/15 after adjusting for confounders (aOR = 1.42 [95% CI: 1.20-1.69]). Conclusions: In general, residential crowding did not change from 2004 to 2014; however, there seems to be a small upsurge for children with low-educated parents, living in urban areas over the years. A statistically significant association between crowding and cough was only found in the survey from 2014/15

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Residential crowding and asthma in preschool children, a cross-sectional study.

BACKGROUND: Asthma is the most common chronic disease in children, and associations with crowding have been reported. The aim of this study was to explore possible associations of crowding with asthma in children. METHODS: Seven cross-sectional surveys with preschool children were conducted within the framework of the health monitoring units in Bavaria, Germany, from 2004 to 2014. Residential crowding was defined as habitation of more than one person per room or less than 20m2 living space per person. Logistic regression models examined temporal changes in crowding, applying the first survey as reference. The relationship between crowding and physician-diagnosed asthma, asthma defined by the International Study of Asthma and Allergies in Childhood (ISAAC) and asthma symptoms were analyzed. RESULTS: Analyzing temporal changes of crowding rates did not reveal any differences over the years. However, the stratified descriptive analysis indicated a crowding increase in time in urban households where parents had a low education level (47.9% in 2004/05, 55.8% in 2014/15). No association was found between crowding and the variables "physician-diagnosed asthma" in 2014/15, "asthma defined by ISAAC" in 2014/15, or "wheezing" in 2014/15. A positive association with cough was identified in 2014/15 after adjusting for confounders (aOR=1.42 [95% CI: 1.20-1.69]). CONCLUSIONS: In general, residential crowding did not change from 2004 to 2014; however, there seems to be a small upsurge for children with low-educated parents, living in urban areas over the years. A statistically significant association between crowding and cough was only found in the survey from 2014/15.

Bivariate and multivariate analyses of the correlations between stability of the erythrocyte membrane, serum lipids and hematological variables.

The observation that the fluidity must remain within a critical interval, outside which the stability and functionality of the cell tends to decrease, shows that stability, fluidity and function are related and that the measure of erythrocyte stability allows inferences about the fluidity or functionality of these cells. This study determined the biochemical and hematological variables that are directly or indirectly related to erythrocyte stability in a population of 71 volunteers. Data were evaluated by bivariate and multivariate analysis. The erythrocyte stability showed a greater association with hematological variables than the biochemical variables. The RDW stands out for its strong correlation with the stability of erythrocyte membrane, without being heavily influenced by other factors. Regarding the biochemical variables, the erythrocyte stability was more sensitive to LDL-C. Erythrocyte stability was significantly associated with RDW and LDL-C. Thus, the level of LDL-C is a consistent link between stability and functionality, suggesting that a measure of stability could be more one indirect parameter for assessing the risk of degenerative processes associated with high levels of LDL-C.

Hypoxia inhibits the peroxisome proliferator-activated receptor alpha/retinoid X receptor gene regulatory pathway in cardiac myocytes: a mechanism for O2-dependent modulation of mitochondrial fatty acid oxidation.

Hypoxia triggers a cascade of cellular energy metabolic responses including a decrease in mitochondrial oxidative flux. To characterize gene regulatory mechanisms by which mitochondrial fatty acid oxidative capacity is diminished in response to hypoxia, cardiac myocytes in culture were exposed to long-chain fatty acids (LCFA) under normoxic or hypoxic conditions. Hypoxia prevented the known LCFA-induced accumulation of mRNA encoding muscle carnitine palmitoyltransferase I (M-CPT I), an enzyme that catalyzes the rate-limiting step in mitochondrial fatty acid oxidation (FAO). Under hypoxic conditions, myocytes exhibited significant accumulation of intracellular neutral lipid consistent with reduced CPT I activity and diminished FAO capacity. Transient transfection experiments demonstrated that the hypoxia-mediated blunting of M-CPT I gene expression occurs at the transcriptional level, is localized to an LCFA/peroxisome proliferator-activated receptor alpha (PPARalpha)/retinoid X receptor (RXR) response element within the M-CPT I gene promoter, and is PPARalpha-dependent. DNA-protein binding studies demonstrated that exposure to hypoxia reduces PPARalpha/RXR binding activity. Immunoblotting studies demonstrated that whereas hypoxia had no effect on nuclear levels of PPARalpha protein, nuclear and cellular RXRalpha levels were reduced. Hypoxia also diminished the 9-cis-retinoic acid-mediated activation of a reporter containing an RXR homodimer response element. These results demonstrate that hypoxia deactivates PPARalpha by reducing the availability of its obligate partner RXR.

Two-stage glucocorticoid induction of CYP3A23 through both the glucocorticoid and pregnane X receptors.

Glucocorticoid inducibility of the CYP3A23 gene is conferred by a multisite unit comprising binding sites for several members of the nuclear receptor superfamily of transcription factors, including the chicken ovalbumin upstream promoter-transcription factor COUP-TF, pregnane X receptor (PXR), and hepatocyte nuclear factor 4 (HNF-4). The presence of three binding sites, each of which interacts with more than one factor, contributes to the complexity of the CYP3A23 glucocorticoid-responsive region. Despite the glucocorticoid sensitivity of this gene, direct binding of ligand-activated glucocorticoid receptor (GR) to the CYP3A23 dexamethasone-responsive region (DexRE) is not required for induction. This study demonstrates that DexRE-2 is the key element within the CYP3A23 proximal promoter, conferring ligand sensitivity via its interaction with the PXR/RXRalpha heterodimer. The DexRE-1 and HNF-4 sites are not ligand-responsive, but are essential accessory elements required for full promoter inducibility. In addition to ligand-mediated activation of PXR, the overall induction response involves a GR-mediated stimulation of PXR and RXRalpha expression. Hence, the induction pathway can be divided into two stages. In stage one, maximal induction requires a GR-dependent increase in PXR and RXRalpha expression, and stage two is characterized by direct transcriptional activation of CYP3A23, which is dependent on ligand-activated PXR as well as accessory factors bound at the DexRE-1 and HNF-4 sites. Because multiple proteins bind at each element within the glucocorticoid-responsive region, factors not contributing to ligand responsiveness, such as chicken ovalbumin upstream promoter-transcription factor, may modulate the response through competitive interactions.

The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in the transcriptional control of genes encoding mitochondrial fatty acid beta-oxidation (FAO) enzymes. In this study we sought to determine whether the recently identified PPAR gamma coactivator 1 (PGC-1) is capable of coactivating PPARalpha in the transcriptional control of genes encoding FAO enzymes. Mammalian cell cotransfection experiments demonstrated that PGC-1 enhanced PPARalpha-mediated transcriptional activation of reporter plasmids containing PPARalpha target elements. PGC-1 also enhanced the transactivation activity of a PPARalpha-Gal4 DNA binding domain fusion protein. Retroviral vector-mediated expression studies performed in 3T3-L1 cells demonstrated that PPARalpha and PGC-1 cooperatively induced the expression of PPARalpha target genes and increased cellular palmitate oxidation rates. Glutathione S-transferase "pulldown" studies revealed that in contrast to the previously reported ligand-independent interaction with PPARgamma, PGC-1 binds PPARalpha in a ligand-influenced manner. Protein-protein interaction studies and mammalian cell hybrid experiments demonstrated that the PGC-1-PPARalpha interaction involves an LXXLL domain in PGC-1 and the PPARalpha AF2 region, consistent with the observed ligand influence. Last, the PGC-1 transactivation domain was mapped to within the NH(2)-terminal 120 amino acids of the PGC-1 molecule, a region distinct from the PPARalpha interacting domains. These results identify PGC-1 as a coactivator of PPARalpha in the transcriptional control of mitochondrial FAO capacity, define separable PPARalpha interaction and transactivation domains within the PGC-1 molecule, and demonstrate that certain features of the PPARalpha-PGC-1 interaction are distinct from that of PPARgamma-PGC-1.

Differential glucocorticoid responses of CYP3A23 and CYP3A2 are mediated by selective binding of orphan nuclear receptors.

CYP3A2 and CYP3A23 are two cytochrome P450 genes in rat that are differentially regulated in both their constitutive activities and their responsiveness to glucocorticoids, the prototypic CYP3A inducers. CYP3A2 displays 20-25% of the response to glucocorticoids as CYP3A23 despite extensive sequence homology in their 5'-regulatory regions. Promoter deletion analyses revealed that the CYP3A2 -57 to -168 region, homologous to the CYP3A23 dexamethasone-responsive region, mediated its low level activation. When this region was analyzed by DNase I footprinting, three binding sites were shown to correspond to the functional elements described for CYP3A23: DexRE-1, DexRE-2, and Site A (J. M. Huss and C. B. Kasper (1998) J. Biol. Chem. 273: 16155-16162). The CYP3A2 DexRE-2 and Site A elements bear two mismatches each from the CYP3A23 elements but displayed similar binding patterns in footprinting and gel-shift analyses as their CYP3A23 counterparts. The region containing 3A2DexRE-1 has six mismatches and displayed unique footprinting and gel-shift patterns compared to 3A23DexRE-1. Functional assays revealed that four mismatches within the DexRE-1 and DexRE-2 elements accounted for the differential inducibility of the two isoforms. We propose that the reduced responsiveness of CYP3A2 is the result of preferential binding of COUP-TF at the CYP3A2 DexRE-1 site. In contrast, CYP3A23 DexRE-1 associates with an accessory factor(s) that acts in concert with downstream sites to mediate the strong glucocorticoid induction response observed for CYP3A23. Site A mismatches did not influence induction magnitude but were responsible for basal activity differences. Higher CYP3A23 basal activity appears to be due to an E-box in 3A23SiteA that interacts with USF1, a ubiquitous bHLH/leucine zipper transcription factor. This site is disrupted in the corresponding 3A2SiteA. Hence, 4 nucleotide mismatches within two elements account for the difference in glucocorticoid induction, and a single mismatch is responsible for the fivefold difference in the basal activities of CYP3A2 and CYP3A23.

Nuclear receptor involvement in the regulation of rat cytochrome P450 3A23 expression.

Many genes of the cytochrome P450 3A (CYP3A) subfamily, including several human and rat isoforms, are inducible by glucocorticoids. In the rat CYP3A23 gene, a 110-base pair segment of the proximal 5'-flanking region mediates dexamethasone activation. Three binding sites (DexRE-1, DexRE-2, and Site A), identified by DNase I footprinting analysis, were characterized for their relative contribution to both basal activity and dexamethasone inducibility. Site-directed mutagenesis of DexRE-1 (-144 to -169) and DexRE-2 (-118 to -136) demonstrated that each contained a core imperfect AGGTCA direct repeat, which comprised a consensus nuclear receptor binding site, and was essential for dexamethasone responsiveness but was not required for basal activity. Competition gel shift and supershift analyses revealed that both sites can bind the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor. Site A (-85 to -110) was shown to be important for both basal activity and dexamethasone responsiveness. Point mutants displayed a reduced (2-3-fold) induction response, compared with 15-fold for wild-type, which was accompanied by a 40-60% drop in basal activity. Site A was shown to bind the liver-enriched nuclear receptor hepatocyte nuclear factor 4. Our studies demonstrate that the mechanism mediating glucocorticoid-inducible transcriptional activity of CYP3A23 involves multiple binding sites for members of the nuclear receptor superfamily.

Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif.

Elements responsible for dexamethasone responsiveness of CYP3A23, a major glucocorticoid-inducible member of the CYP3A gene family, have been identified. DNase I footprint analysis of the proximal promoter region revealed three protected sites (sites A, B, and C) within the sequence defined by -167 to -60. Mutational analysis demonstrated that both sites B and C were necessary for maximum glucocorticoid responsiveness and functioned in a cooperative manner. Interestingly, neither site contained a glucocorticoid responsive element. Embedded in site C was an imperfect direct repeat (5'-AACTCAAAGGAGGTCA-3'), showing homology to an AGGTCA steroid receptor motif, typically recognized by the estrogen receptor family, while site B contained an ATGAACT direct repeat; these core sequences were designated dexamethasone response elements 1 and 2 (DexRE-1 and -2), respectively. Neither element has previously been associated with a glucocorticoid-activated transcriptional response. Conversion of the DexRE-1 to either a perfect thyroid hormone or vitamin D3 responsive element further enhanced induction by dexamethasone. Gel-shift analysis demonstrated that glucocorticoid receptor did not associate with either DexRE-1 or -2; hence, glucocorticoid receptor does not directly mediate glucocorticoid induction of CYP3A23. These unusual features suggest an alternate pathway through which glucocorticoids exert their effects.

Age-specific neurotoxicity in the rat associated with NMDA receptor blockade: potential relevance to schizophrenia?

Agents that block the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor induce a schizophrenialike psychosis in adult humans and injure or kill neurons in several corticolimbic regions of the adult rat brain. Susceptibility to the psychotomimetic effects of the NMDA antagonist, ketamine is minimal or absent in children and becomes maximal in early adulthood. We examined the sensitivity of rats at various ages to the neurotoxic effects of the powerful NMDA antagonist, MK-801. Vulnerability was found to be age dependent, having onset at approximately puberty (45 days of age) and becoming maximal in early adulthood. This age-dependency profile (onset of susceptibility in late adolescence) in the rat is similar to that for ketamine-induced psychosis or schizophrenia in humans. These findings suggest that NMDA receptor hypofunction, the mechanism underlying the neurotoxic and psychotomimetic actions of NMDA antagonists, may also play a role in schizophrenia.

Quisqualate injection into the nucleus basalis magnocellularis produces seizure-related brain damage that is prevented by MK-801.

Quisqualate (Quis) and other excitotoxins such as ibotenate and N-methyl aspartate, have been used to destroy neurons in the area of the nucleus basalis magnocellularis (NBM) in order to study the relationship between loss of cholinergic neurons in the basal forebrain and various behavioral deficits, including learning and memory impairments. The results of several studies suggest that although Quis NBM lesions may produce greater depletions in cortical choline acetyltransferase levels than ibotenate lesions, the learning/memory deficits tend to be milder following Quis lesions. In these studies, it was often assumed that the lesions induced by Quis were restricted to the local vicinity of the injection. However, in the present study, we found that an injection of Quis into the NBM/substantia inominata (SI) region often induces limbic seizures and disseminated brain damage. Specifically, we found that an injection of Quis into the NBM/SI area of female rats at a dose (120 nmol) used by others in previous behavioral studies produced massive damage in areas distant from the lesion site, particularly in the amygdala and piriform cortex. This disseminated damage occurred in 50% of the rats treated with Quis, was typically more severe than damage at the injection site, and was often accompanied by equally severe "mirror" lesions in the contralateral amygdala and piriform cortex. Injecting rats with MK-801 (1 mg/kg) 30 min before the Quis injection protected against the disseminated damage. These data underscore the need for careful histological evaluation of excitotoxic lesions and for caution in interpreting the relationship between altered transmitter markers and learning/memory impairment seen following these lesions.

Postnatal muscle fibre histochemistry in the rat.

Histochemical techniques were used to study the postnatal muscle fibre differentiation patterns in the plantaris and soleus muscles of male Sprague-Dawley rats. Nine groups of animals (n = 6/group) were killed at 1, 6, 11, 16, 21, 26, 31, 36, and 140 days of age. Serial transverse sections of the two muscles were stained with H & E, NADH-D, and myofibrillar ATPase with acid (pH 4.35) or alkali (pH 10.4) preincubation. In each of the age groups, all available fibres across the muscle sections were classified. Obtained data show that fibre types are basically undifferentiated at birth in both muscles. In the plantaris muscle there are about 99% type IIA and less than 1% type I fibres at 6 days of age. Type IIB fibres can be identified at 11 days of age. There are increases in the percentages of type I fibres (from 0.7% to 3.5%) and type IIB fibres (from 1.1% to 6.5%) between 6 and 11 days and between 16 and 21 days respectively. By 36 days of age the relative numbers of type IIA, IIB, and type I fibres in the plantaris are approximately 80%, 14%, and 6%, respectively. A gradual change in fibre-type composition continues until it becomes 47% for type IIA, 43% for type IIB, and 10% for type I at 140 days of age. In the soleus muscle there are approximately 73% type IIA and 26% type I fibres at both 6 and 11 days of age. However, type IIA fibres decrease to 44% and type I fibres increase to 56% at 16 days of age. This rapid shift in fibre composition continues up to 31 days of age when the distribution becomes 25% for type IIA and 74% for type I fibres. Thereafter, the differentiation rate is much slower. At 140 days of age, there are 17% type IIA and 83% type I fibres in the soleus muscle. The results of this study show that the fibre populations in the plantaris and soleus muscles of the rat undergo a postnatal differentiation process. In both muscles the adult fibre population is established by 140 days of age. Although relatively rapid increases of type I and type IIB fibres occur in the plantaris during the second and third weeks of life, differentiation in that muscle appears to be an essentially continuous process. There is a notable shift in the fibre composition of the soleus muscle during the second postnatal week. Differences between the patterns of differentiation in the two muscles are apparent.

LH and FSH responsiveness to intravenous gonadotropin-releasing hormone (GnRH) in children with hypothalamic or pituitary disorders: lack of effect of replacement therapy with human growth hormone.

To evaluate the diagnostic and prognostic usefulness of the GnRH test, gonadotropin responses to iv GnRH (Parke-Davis) were determined in 82 young patients (2,5 mo.-21 yr.) and 6 normal men. After extensive evaluation, 40 patients (31 boys and 9 girls) were considered "endocrinologically normal." Repeat tests were performed in 17 patients at 6-12 mo. intervals. Nine patients with presumed isolated hGH deficiency and 3 patients with multiple pituitary deficiencies were studied before and at the end of 12 mo. of hGH therapy. Serial blood samples were obtained before and after an iv bolus injection of GnRH (2.5 mug/kg, 74 tests, or 10 mug/m2, 36 tests). LH and FSH were determined by radioimmunoassay. Maximum concentration, maximum increment (deltamax), and response area were compared with degree of skeletal maturation to evaluate responses. Clinically, the most useful determination was the deltamax LH. All "normal" children with bone ages greater than 12 yr had LH responses in or slightly above the range of the values of the 6 normal men: deltamax LH, 39 +/- 8 (SE) mIU/ml; range 13-57. Severely blunted or absent responses were seen in 14/15 patients (bone ages 3 mo.-14 yr.) with multiple pituitary deficiencies. Boys with isolated hGH deficiency and bone ages of less than 10 yr had significantly lower responses than "short normal" boys with similar skeletal maturation: deltamax LH, 4.8 +/- 0.9 vs 8 +/- 1.3 mIU/ml, P is less than .05. Although the mean growth velocity of hGH-treated children increased from 3.2 to 8.9 cm/yr, LH and FSH responses were unchanged. These studies indicate that 1) children with idiopathic hypopituitarism (including those with presumed isolated hGH deficiency) have significantly decreased responsiveness to GnRH which does not respond to 6 to 12 months of hGH treatment; and 2) decreased responsiveness to GnRH in patients with bone ages of greater than 12 yr is presumptive evidence of gonadotropin deficiency.