Spectrum of pathogens of in-patient children and youths with community acquired pneumonia: a 3 year survey of a community hospital in Vienna, Austria.

BACKGROUND: No actual data are available on the epidemiology and morbidity of community acquired pneumonia (CAP) in youths and children in Vienna, Austria. OBJECTIVE: The objective was to determine the epidemiology of CAP and morbidity of children hospitalized due to CAP in a tertiary care facility. METHODS: During three winter seasons youths and children hospitalized due to CAP were enrolled. Testing for viral and bacterial pathogens of pneumonia was performed in a routine clinical setting. Blood cultures were performed; respiratory viruses, Mycoplasma pneumoniae and Chlamydia pneumoniae were searched for by an established Real Time polymerase chain reaction (PCR) panel. Clinical signs and indices of inflammation were documented. RESULTS: Out of 279 children and youths with CAP a causative agent could be detected in 190 (68 %). Viruses and bacteria were diagnosed in 107 (57 %) and 58 patients (30 %), respectively. Co-infection was found in 20 patients (10 %), Mycoplasma pneumoniae or Clamydia pneumoniae in 16 cases (8 %). In seven patients blood cultures were positive. C-reactive protein (CRP) was significantly higher in children with positive Streptococcus pneumoniae antigen (SPAG) than with viral infection and/or co-infection. Clinical parameters showed no statistically significant differences. C. pneumoniae and M. pneumoniae were only diagnosed in children and youths with 5 years and older. CONCLUSIONS: Testing for pathogens in CAP in clinical routine achieves a high recovery rate. Blood cultures are rarely helpful, but the molecular testing for viruses seemed to be helpful to establish the diagnosis.

Detection of Chlamydophila pneumoniae in cats with conjunctivitis.

OBJECTIVE: To determine the presence of chlamydial species including recently described chlamydial agents as well as the human pathogen Chlamydophila pneumoniae in feline conjunctivitis. ANIMAL STUDIED: Twenty five cats without and 49 cats with conjunctivitis were tested for chlamydia using a Chlamydiaceae real time (RT) PCR (targeting the 23S rRNA gene sequence), a Chlamydiales PCR (targeting the 16S rRNA gene sequence), and cell culture. The PCR products of all positive samples were sequenced and subsequently analyzed using a basic local alignment search tool search. RESULTS: Chlamydiaceae RT PCR and subsequent sequence analyses identified C. pneumoniae in five cats in the conjunctivitis group. The presence of Chlamydophila felis was shown in two cats with conjunctivitis. Chlamydiae related to uncultured members of Chlamydiales were detected in three conjunctivitis cases and in one cat without clinical symptoms. CONCLUSION: This study detects for the first time, the known human pathogen C. pneumoniae in feline conjunctivitis cases using Chlamydiaceae RT PCR and sequence analyses.

Role of Chlamydophila pneumoniae in children hospitalized for community-acquired pneumonia in Vienna, Austria.

BACKGROUND: No recent Austrian data are available on the epidemiology and morbidity of Chlamydophila pneumoniae (C. pneumoniae) as causative agent in youths and children hospitalized with community-acquired pneumonia (CAP). These data would serve as a rationale for empiric antimicrobial therapy. METHODS: During winter months 2006/7 112 immunocompetent youths and children between 2 months and 18 years hospitalized consecutively with CAP were prospectively enrolled. Microbiological detection of conventional bacteria, Mycoplasma pneumoniae as well as respiratory viruses was performed, and clinical signs as well as indices of inflammation were documented. Diagnosis of C. pneumoniae was performed by means of species-specific real-time PCR as well as cell culture from respiratory secretions. RESULTS: PCR for C. pneumoniae was performed in 60 patients. In 4 cases (6.7%) C. pneumoniae was found as causative agent. In 1 case C. pneumoniae could be confirmed by cell-culture. All children with C. pneumonia were > or =5 years of age. All but 1 child received clarithromycin together with cefuroxime. The number of days with fever >38,5 degrees C was between 1 and 7, the number of days in hospital was between 4 and 7, all children recovered completely. CONCLUSION: C. pneumoniae was found in 6.7% of children and youths hospitalized with CAP in a community hospital during the winter months 2006/07. Children and youths with lower respiratory infections should further be tested for C. pneumoniae to elucidate the actual epidemiological situation and to guide empiric antimicrobiotic therapy.

[18F]FETO: metabolic considerations.

PURPOSE: 11beta-Hydroxylase is a key enzyme in the biosynthesis of adrenocortical steroid hormones and is a suitable target for the imaging of the adrenal cortex. [(11)C]Metomidate (MTO), [(11)C]etomidate (ETO) and desethyl-[(18)F]fluoroethyl-etomidate (FETO) are potent inhibitors of this enzyme and are used for PET imaging of adrenocortical pathologies. The aims of this study were (1) to evaluate and compare the metabolic stability of MTO, ETO and FETO against esterases and (2) to investigate the metabolic pattern of FETO in vivo. METHODS: In vitro assays were performed using different concentrations of MTO, ETO and FETO with constant concentrations of carboxylesterase. Human in vivo studies were performed with human blood samples drawn from the cubital vein. After sample clean-up, the serum was analysed by HPLC methods. RESULTS: In vitro assays showed Michaelis-Menten constants of 115.1 mumol for FETO, 162.0 mumol for MTO and 168.6 mumol for ETO. Limiting velocities were 1.54 mumol/min (FETO), 1.47 mumol/min (MTO) and 1.35 mumol/min (ETO). This implies insignificantly decreased esterase stability of FETO compared with MTO and ETO. In vivo investigations showed a rapid metabolisation of FETO within the first 10 min (2 min: 91.41%+/-6.44%, n=6; 10 min: 23.78%+/-5.54%, n=4) followed by a smooth decrease in FETO from 20 to 90 min (20 min: 11.23%+/-3.79% n=4; 90 min: 3.68%+/-3.65%, n=4). Recovery rate was 61.43%+/-3.19% (n=12). CONCLUSION: In vitro experiments demonstrated that FETO stability against esterases is comparable to that of ETO and MTO. The metabolic profile showed that FETO kinetics in humans are fast.

Biological evaluation of 2'-[18F]fluoroflumazenil ([18F]FFMZ), a potential GABA receptor ligand for PET.

[(11)C]Flumazenil, a highly selective benzodiazepine antagonist is the most extensively used GABA(A) ligand for PET so far. To overcome half life disadvantages of (11)C a [(18)F]-labeled flumazenil derivative, 2'-[(18)F]fluoroflumazenil (FFMZ) was developed and biologically evaluated with respect to the GABA(A) receptor. Organ with the highest uptake was the pituitary gland. Brain uptake was high and followed the order cortex>thalamus>cerebellum>rest brain. Fluoroflumazenil displaced [(3)H]flumazenil binding from membrane GABA(A) receptors with an IC(50)value (3.5 nM) comparable to that of Flumazenil (2.8 nM). The presented data confirm the potential of [(18)F]FFMZ for PET imaging of the GABA-ergic system.

In vivo and in vitro evaluation of [18F]FETO with respect to the adrenocortical and GABAergic system in rats.

11beta-Hydroxylase (CYP11B1, P45011beta) plays an important role in the biosynthesis of cortisol and aldosterone and has been shown to be a good target for the in vivo imaging of adrenocortical incidentalomas in nuclear medicine. [11C]Metomidate (MTO), a potent inhibitor of this enzyme, is used for positron emission tomography (PET) imaging of adrenocortical pathology. The synthesis of (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid 2-[18F]fluoroethylester (FETO), a close analogue to MTO and etomidate (ETO), has been presented recently, and the present investigation aimed to characterise the in vivo distribution of FETO. Since ETO is a well-known anaesthetic drug acting via the GABAergic system, the interaction of FETO with GABAA receptors was also evaluated. Eighteen male Sprague-Dawley rats were injected with 1.73-3.06 MBq of FETO into a tail vein after venodilatation in a 40 degrees C water bath. Rats were sacrificed by exsanguination from the abdominal aorta under deep ether anaesthesia after 10 (n=6), 30 (n=6) or 60 min (n=6); organs were removed, weighed and counted. For binding experiments, rat cerebellar membranes were incubated for 90 min at 4 degrees C in TC-50 buffer, 150 mM NaCl and 2 nM of [3H]flunitrazepam in the absence or presence of 10 microM diazepam or various concentrations of ETO, MTO and FETO. In vivo evaluation evinced very high uptake in the adrenal glands (7.52%+/-1.19% ID/g at 30 min), followed by lung (1.18%+/-0.19% ID/g, 10 min), liver (0.59%+/-0.13% ID/g, 10 min) and duodenum (0.7%+/-0.29% ID/g, 60 min). No defluorination nor fluoroethyl-ester cleavage was observed. When brain regions were compared with the thalamus (the reference region), highest relative uptake was seen in the cortex (2.34), followed by "rest brain" (2.13) and cerebellum (1.96). FETO and ETO were able to increase the binding of [3H]flunitrazepam with similar potencies and to a comparable extent. It is concluded that FETO shows characteristics suitable for the imaging of adrenocortical pathology with PET. Binding experiments on GABA receptors demonstrate a comparable effect of FETO and ETO. Hence, FETO possibly could also be used to elucidate the function, dynamics and kinetics of narcotic drugs with PET.

Saccharomyces cerevisiae PIP2 mediating oleic acid induction and peroxisome proliferation is regulated by Adr1p and Pip2p-Oaf1p.

Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively. The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE. Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2. Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type. In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type. Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells. However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control. This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid. Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.

Identification of an amino acid sequence within GABA(A) receptor beta3 subunits that is important for receptor assembly.

GABA(A) receptors are chloride ion channels that can be opened by GABA, the most important inhibitory transmitter in the CNS. In the mammalian brain the majority of these pentameric receptors is composed of two alpha, two beta and one gamma subunit. To achieve the correct order of subunits around the pore, each subunit must form specific contacts via its plus (+) and minus (-) side. To identify a sequence on the beta3 subunit important for assembly, we generated various full-length or truncated chimeric beta3 constructs and investigated their ability to assemble with alpha1 and gamma2 subunits. It was demonstrated that replacement of the sequence beta3(76-89) by the homologous alpha1 sequence impaired assembly with alpha1 but not with gamma2 subunits in alpha1beta3gamma2-GABA(A) receptors. Other experiments indicated that assembly was impaired via the beta3(-) side of the chimeric subunit. Within the sequence beta3(76-89) the sequence beta3(85-89) seemed to be of primary importance for assembly with alpha1 subunits. A comparison with the structure of the acetylcholine-binding protein supports the conclusion that the sequence beta3(85-89) is located at the beta3(-) side and indicates that it contains amino acid residues that might directly interact with the (+) side of the neighbouring alpha1 subunit.