Sub-inhibitory concentrations of vancomycin prevent quinolone-resistance in a penicillin-resistant isolate of Streptococcus pneumoniae.

BACKGROUND: The continuous spread of penicillin-resistant pneumococci represents a permanent threat in the treatment of pneumococcal infections, especially when strains show additional resistance to quinolones. The main objective of this study was to determine a treatment modality impeding the emergence of quinolone resistance. RESULTS: Exposure of a penicillin-resistant pneumococcus to increasing concentrations of trovafloxacin or ciprofloxacin selected for mutants resistant to these drugs. In the presence of sub-inhibitory concentrations of vancomycin, development of trovafloxacin-resistance and high-level ciprofloxacin-resistance were prevented. CONCLUSIONS: Considering the risk of quinolone-resistance in pneumococci, the observation might be of clinical importance.

Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to suinhibitory antibiotic concentrations

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Growth, yield and secondary metabolite production of Drosera species cultivated in peat beds in Finland

Tiivistelmä

Retinaldehyde: more than meets the eye.

Retinaldehyde, an intermediate metabolite between vitamin A and retinoic acid, is present at biologically active concentrations in fat tissue, where it antagonizes PPAR- activity, inhibiting adipogenesis and improving insulin sensitivity.

LDL-cholesterol concentrations: a genome-wide association study.

BACKGROUND: LDL cholesterol has a causal role in the development of cardiovascular disease. Improved understanding of the biological mechanisms that underlie the metabolism and regulation of LDL cholesterol might help to identify novel therapeutic targets. We therefore did a genome-wide association study of LDL-cholesterol concentrations. METHODS: We used genome-wide association data from up to 11,685 participants with measures of circulating LDL-cholesterol concentrations across five studies, including data for 293 461 autosomal single nucleotide polymorphisms (SNPs) with a minor allele frequency of 5% or more that passed our quality control criteria. We also used data from a second genome-wide array in up to 4337 participants from three of these five studies, with data for 290,140 SNPs. We did replication studies in two independent populations consisting of up to 4979 participants. Statistical approaches, including meta-analysis and linkage disequilibrium plots, were used to refine association signals; we analysed pooled data from all seven populations to determine the effect of each SNP on variations in circulating LDL-cholesterol concentrations. FINDINGS: In our initial scan, we found two SNPs (rs599839 [p=1.7x10(-15)] and rs4970834 [p=3.0x10(-11)]) that showed genome-wide statistical association with LDL cholesterol at chromosomal locus 1p13.3. The second genome screen found a third statistically associated SNP at the same locus (rs646776 [p=4.3x10(-9)]). Meta-analysis of data from all studies showed an association of SNPs rs599839 (combined p=1.2x10(-33)) and rs646776 (p=4.8x10(-20)) with LDL-cholesterol concentrations. SNPs rs599839 and rs646776 both explained around 1% of the variation in circulating LDL-cholesterol concentrations and were associated with about 15% of an SD change in LDL cholesterol per allele, assuming an SD of 1 mmol/L. INTERPRETATION: We found evidence for a novel locus for LDL cholesterol on chromosome 1p13.3. These results potentially provide insight into the biological mechanisms that underlie the regulation of LDL cholesterol and might help in the discovery of novel therapeutic targets for cardiovascular disease.

Cytochrome P450 and ABCB1 genetics: association with quetiapine and norquetiapine plasma and cerebrospinal fluid concentrations and with clinical response in patients suffering from schizophrenia. A pilot study.

Variability in response to atypical antipsychotic drugs is due to genetic and environmental factors. Cytochrome P450 (CYP) isoforms are implicated in the metabolism of drugs, while the P-glycoprotein transporter (P-gp), encoded by the ABCB1 gene, may influence both the blood and brain drug concentrations. This study aimed to identify the possible associations of CYP and ABCB1 genetic polymorphisms with quetiapine and norquetiapine plasma and cerebrospinal fluid (CSF) concentrations and with response to treatment. Twenty-two patients with schizophrenia receiving 600 mg of quetiapine daily were genotyped for four CYP isoforms and ABCB1 polymorphisms. Quetiapine and norquetiapine peak plasma and CSF concentrations were measured after 4 weeks of treatment. Stepwise multiple regression analysis revealed that ABCB1 3435C > T (rs1045642), 2677G > T (rs2032582) and 1236C > T (rs1128503) polymorphisms predicted plasma quetiapine concentrations, explaining 41% of the variability (p = 0.001). Furthermore, the ABCB1 polymorphisms predicted 48% (p = 0.024) of the variability of the Δ PANSS total score, with the non-carriers of the 3435TT showing higher changes in the score. These results suggest that ABCB1 genetic polymorphisms may be a predictive marker of quetiapine treatment in schizophrenia.

Effect of age and gender on citalopram and desmethylcitalopram steady-state plasma concentrations in adults and elderly depressed patients.

The effect of aging on steady-state plasma concentrations of citalopram (CIT) and desmethylcitalopram (DCIT) was investigated in 128 depressive patients treated with 10-80 mg/day CIT. They were separated into three groups, with age up to 64 years (mean age+/-S.D.: 47+/-12 years; n=48), between 65 and 79 years (72+/-1 years; n=57), and from 80 years or older (84+/-1 years; n=23). Body mass index (BMI), renal and hepatic functions were similar in the three groups. A large interindividual variability of plasma levels of CIT (16-fold) and DCIT (12-fold) was measured for a given dose. The mean plasma levels of CIT corrected for a 20 mg daily dose were 55% higher in the very elderly (>=80 years) patients (65+/-30 ng/ml; p<0.001) and 38% higher in the elderly (65-79 years) patients (58+/-24 ng/ml; p<0.001) when compared to the adult patients (42+/-17 ng/ml). DCIT mean plasma level was 38% higher (p<0.05) in the group of very elderly patients (22+/-10 ng/ml) when compared to the adult patients (16+/-9 ng/ml). As a consequence, the mean plasma concentration of CIT+DCIT was 48% higher in the very elderly patients (86+/-36 ng/ml; p<0.001) and 33% higher in the elderly patients (77+/-28 ng/ml; p<0.001) when compared to the adult patients (58+/-21 ng/ml). Age correlated significantly with CIT (r=0.43, p<0.001), DCIT (r=0.28, p<0.01), and CIT+DCIT plasma levels (r=0.44, p<0.001), and thus accounts for 18% of the variability of CIT plasma levels, with no influence of gender. The recommended dose reduction of CIT in elderly patients seems therefore justified.

Metal concentrations in soils around the copper smelter and surrounding industrial complex of Port Kembla, NSW, Australia

Anthropogenic emissions of metals from sources such as smelters are an international problem, but there is limited published information on emissions from Australian smelters. The objective of this study was to investigate the regional distribution of heavy metals in soils in the vicinity of the industrial complex of Port Kembla, NSW, Australia, which comprises a copper smelter, steelworks and associated industries. Soil samples (n=25) were collected at the depths of 0-5 and 5-20 cm, air dried and sieved to < 2 mm. Aqua regia extractable amounts of As, Cr, Cu, Ph and Zn were analysed by inductively coupled plasma mass spectrometry (lCP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). Outliers were identified from background levels by statistical methods. Mean background levels at a depth of 0-5 cm were estimated at 3.2 mg/kg As, 12 mg/kg Cr, 49 mg/kg Cu, 20 mg/kg Ph and 42 mg/kg Zn. Outliers for elevated As and Cu values were mainly present within 4 km from the Port Kembla industrial complex, but high Ph at two sites and high Zn concentrations were found at six sites up to 23 km from Port Kembla. Chromium concentrations were not anomalous close to the industrial complex. There was no significant difference of metal concentrations at depths of 0-5 and 5-20 cm, except for Ph and Zn. Copper and As concentrations in the soils are probably related to the concentrations in the parent rock. From this investigation, the extent of the contamination emanating from the Port Kembla industrial complex is limited to 1-13 km, but most likely <4 km, depending on the element; the contamination at the greater distance may not originate from the industrial complex. (C) 2003 Elsevier B.V. All rights reserved.

A phase I pharmacokinetic study of hypoxic abdominal stop-flow perfusion with gemcitabine in patients with advanced pancreatic cancer and refractory malignant ascites

PURPOSE: As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n = 8, and cholangiocarcinoma of the liver, n = 1). EXPERIMENTAL DESIGN: Gemcitabine at 500, 750 and 1,125 mg/m(2) was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2',2'-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20 min of stop-flow perfusion, and in peripheral plasma for 420 min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH </= 7.2, pO(2) nadir ratio </=0.70 or pCO(2) peak ratio >/=1.35. The tolerability of this procedure was also assessed. RESULTS: Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20 min of stop-flow perfusion, with C (max) levels in the abdominal circuit of 246 (+/-37%), 2,039 (+/-77%) and 4,780 (+/-7.3%) mug/ml for the three dose levels 500, 750 and 1,125 mg/m(2), respectively. These C (max) were between 13 (+/-51%) and 290 (+/-12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUC(t0-20), was between 5.5 (+/-43%) and 200 (+/-66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P < 0.01 for C (max) and AUC(t0-20), both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P < 0.05) was found between systemic C (max) of gemcitabine and the nadir of both leucocytes and neutrophils. CONCLUSIONS: Regional exposure to gemcitabine-the current standard drug for advanced adenocarcinoma of the pancreas-can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125 mg/m(2). However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherapy.

Determination of meropenem in plasma and filtrate-dialysate from patients under continuous veno-venous haemodiafiltration by SPE-LC.

Meropenem, a carbapenem antibiotic displaying a broad spectrum of antibacterial activity, is administered in Medical Intensive Care Unit to critically ill patients undergoing continuous veno-venous haemodiafiltration (CVVHDF). However, there are limited data available to substantial rational dosing decisions in this condition. In an attempt to refine our knowledge and propose a rationally designed dosage regimen, we have developed a HPLC method to determine meropenem after solid-phase extraction (SPE) of plasma and dialysate fluids obtained from patients under CVVHDF. The assay comprises the simultaneous measurement of meropenem's open-ring metabolite UK-1a, whose fate has never been studied in CVVHDF patients. The clean-up procedure involved a SPE on C18 cartridge. Matrix components were eliminated with phosphate buffer pH 7.4 followed by 15:85 MeOH-phosphate buffer pH 7.4. Meropenem and UK-1a were subsequently desorbed with MeOH. The eluates were evaporated under nitrogen at room temperature (RT) and reconstituted in phosphate buffer pH 7.4. Separation was performed at RT on a Nucleosil 100-5 microm C18 AB cartridge column (125 x 4 mm I.D.) equipped with a guard column (8 x 4 mm I.D.) with UV-DAD detection set at 208 nm. The mobile phase was 1 ml min(-1), using a step-wise gradient elution program: %MeOH/0.005 M tetrabutylammonium chloride pH 7.4; 10/90-50/50 in 27 min. Over the range of 5-100 microg ml(-1), the regression coefficient of the calibration curves (plasma and dialysate) were >0.998. The absolute extraction recoveries of meropenem and UK-1a in plasma and filtrate-dialysate were stable and ranged from 88-93 to 72-77% for meropenem, and from 95-104 to 75-82% for UK-1a. In plasma and filtrate-dialysate, respectively, the mean intra-assay precision was 4.1 and 2.6% for meropenem and 4.2 and 3.7% for UK-1a. The inter-assay variability was 2.8 and 3.6% for meropenem and 2.3 and 2.8% for UK-1a. The accuracy was satisfactory for both meropenem and UK-1a with deviation never exceeding 9.0% of the nominal concentrations. The stability of meropenem, studied in biological samples left at RT and at +4 degrees C, was satisfactory with < 5% degradation after 1.5 h in blood but reached 22% in filtrate-dialysate samples stored at RT for 8 h, precluding accurate measurements of meropenem excreted unchanged in the filtrate-dialysate left at RT during the CVVHDF procedure. The method reported here enables accurate measurements of meropenem in critically ill patients under CVVHDF, making dosage individualisation possible in such patients. The levels of the metabolite UK-1a encountered in this population of patients were higher than those observed in healthy volunteers but was similar to those observed in patients with renal impairment under hemodialysis.

Serum resistin concentrations and risk of new onset heart failure in older persons: the health, aging, and body composition (Health ABC) study.

OBJECTIVE: Resistin is associated with inflammation and insulin resistance and exerts direct effects on myocardial cells including hypertrophy and altered contraction. We investigated the association of serum resistin concentrations with risk for incident heart failure (HF) in humans. METHODS AND RESULTS: We studied 2902 older persons without prevalent HF (age, 73.6+/-2.9 years; 48.1% men; 58.8% white) enrolled in the Health, Aging, and Body Composition (Health ABC) Study. Correlation between baseline serum resistin concentrations (20.3+/-10.0 ng/mL) and clinical variables, biochemistry panel, markers of inflammation and insulin resistance, adipocytokines, and measures of adiposity was weak (all rho <0.25). During a median follow-up of 9.4 years, 341 participants (11.8%) developed HF. Resistin was strongly associated with risk for incident HF in Cox proportional hazards models controlling for clinical variables, biomarkers, and measures of adiposity (HR, 1.15 per 10.0 ng/mL in adjusted model; 95% CI, 1.05 to 1.27; P=0.003). Results were comparable across sex, race, diabetes mellitus, and prevalent and incident coronary heart disease subgroups. In participants with available left ventricular ejection fraction at HF diagnosis (265 of 341; 77.7%), association of resistin with HF risk was comparable for cases with reduced versus preserved ejection fraction. CONCLUSIONS: Serum resistin concentrations are independently associated with risk for incident HF in older persons.

Increased vulnerability of neurones and glial cells to low concentrations of methylmercury in a prooxidant situation.

Using reaggregating rat brain cell cultures at two different stages of differentiation, we examined the biochemical effects of a 10-day treatment with nanomolar concentrations of methylmercuric chloride (monomethylmercury), in the presence or absence of promoters of hydroxyl radical formation (10 microM copper sulphate plus 100 microM ascorbate). A decrease in total protein content accounted for the general cytotoxicity of these compounds, whereas selective effects were assessed by determining the activities of cell type-specific enzymes. Methylmercury, up to 100 nM, as well as the copper ascorbate mixture, when applied separately, induced no general cytotoxicity, and only slight effects on neuronal parameters. However, when applying 100 nM methylmercury and the copper-ascorbate mixture together, a drastic decrease in neuronal and glial parameters was found. Under these conditions, the content of reactive oxygen species, assessed by 2',7'-dichlorofluorescin oxidation, increased greatly, while the activities of antioxidant enzymes decreased. In the presence of copper and ascorbate, differentiated cultures appeared more resistant than immature ones to low methylmercury concentrations (1-10 mM), but did undergo similar changes in both cell type-specific and antioxidant enzyme activities at 100 nM methylmercury. These results suggest that in prooxidant conditions low doses of mercury can become much more deleterious for the central nervous system.