Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Moving beyond the concept of "primary forest" as a metric of forest environment quality.

The United Nations Food and Agriculture Organization (FAO) has been reporting country-level area in primary forests in its Global Forest Resource Assessment since 2005. The FAO definition of a primary forest (naturally regenerated forest of native species where there are no clearly visible indications of human activities and the ecological processes are not significantly disturbed) is generally accepted as authoritative and is being used in policy making. However, problems with this definition undermine our capacity to obtain globally coherent estimates. In addition, the current reporting on primary forests fails to consider the complementarily of non-primary forests toward the maintenance of ecosystem services. These issues undermine the appropriate tracking of changes in primary and non-primary forests, and the assessment of impacts of such changes on ecosystem services. We present the case for an operational reconsideration of the primary forest concept and discuss how alternatives or supplements might be developed.

Hepatitis C virus and atherosclerosis: A legacy after virologic cure?

Hepatitis C virus (HCV) is a major pathogen with approximately 3% of the world's population (over 170 million) infected. Epidemiological studies have shown HCV is associated with an increased risk of cardiovascular and cerebrovascular mortality as well as peripheral arterial disease. This is despite HCV inducing an ostensibly favourable lipid profile with accompanying low classical risk score for atherosclerosis (AS). We discuss possible factors involved in the aetiopathogenesis of atherosclerosis in chronic HCV and hypothesise that an important mechanism underlying the development of AS is the presence of circulating low-density immune complexes that induce an inflammatory response. We suggest that HCV particles may be inducing an antibody response to lipoproteins present in the lipoviral particles and sub-viral particles - a concept similar to the more general 'autoantibody' response to modified LDL. After virologic cure some AS risk factors will recede but an increase in serum cholesterol could result in progression of early atherosclerotic lesions, leaving a legacy from persistent HCV infection that has clinical and therapeutic implications.

Depressive symptoms in chronic hepatitis C are associated with plasma apolipoprotein E deficiency.

Neuro-psychiatric and cognitive disorders are frequent in patients with chronic hepatitis C (CHC) virus (HCV) infection which adversely impact quality of life, antiviral treatment adherence and outcome. HCV has neurotrophic properties and affects lipid metabolism, essential for cognitive function. We evaluated the relationship of lipid profiles with depression and anxiety symptoms and the effects of 12-weeks of therapy with fluvastatin and omega-3 ethyl esters (n-3 PUFA) in a randomised pilot study of CHC prior non-responders. Participants (n = 60) had fasting lipid profiles and assessment of depression and anxiety symptoms using the Hospital Anxiety and Depression Scale (HADS) questionnaire at each study visit. At screening 26/60 (43 %) had HADS-A score ≥8 and 13/60 (22 %) had HADS-D scores ≥8. Depressed patients had significantly lower apolipoprotein-E concentrations (30 mg/l vs 39 mg/l, P = 0.029) than those without depression and a tendency toward lower total cholesterol (3.8 vs 4.4 mmol/l, P = 0.053). 3 patients discontinued lipid-modifying treatment because of worsening depression. However, there was a small but significant improvement in anxiety symptoms after 12-weeks of high-dose (2-4 g daily) n-3 PUFA. In conclusion, depression in CHC is associated with plasma apoE deficiency. We postulate that apoE deficiency disrupts blood brain barrier integrity to promote HCV infection of the CNS. High-dose n-PUFAs may alleviate anxiety in some CHC patients but the use of lipid lowering therapy must be balanced against risks of worsening depression.

Lipids and HCV.

Chronic hepatitis C virus (HCV) infection is associated with an increase in hepatic steatosis and a decrease in serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL) and apolipoprotein B (apoB), the main protein constituent of LDL and very low-density lipoprotein (VLDL). These changes are more marked in HCV genotype 3 infection, and effective treatment results in their reversal. Low lipid levels in HCV infection correlate not only with steatosis and more advanced liver fibrosis but also with non-response to interferon-based therapy. The clinical relevance of disrupted lipid metabolism reflects the fact that lipids play a crucial role in the life cycle of hepatitis C virus. HCV assembly and maturation in hepatocytes depend on microsomal triglyceride transfer protein and apoB in a manner that parallels the formation of VLDL. VLDL production from the liver occurs throughout the day with an estimated 10(18) particles produced every 24 h whilst the estimated hepatitis C virion production rate is 10(12) virions per day. HCV particles in the serum exist as a mixture of complete low-density infectious lipo-viral particles (LVP) and a vast excess of apoB-associated empty nucleocapsid-free sub-viral particles that are complexed with anti-HCV envelope antibodies. Apolipoprotein E (apoE) is also involved in HCV particle morphogenesis and is an essential apolipoprotein for HCV infectivity. ApoE is a critical ligand for the receptor-mediated removal of triglyceride rich lipoprotein (TRL) remnants by the liver. The dynamics of apoB-associated lipoproteins, including HCV-LVP, change post-prandially with an increase in large TRL remnants and very low density HCV-LVP which are rapidly cleared by the liver (at least three HCV receptors are cellular receptors for uptake of TRL remnants). In summary, HCV utilises triglyceride-rich lipoprotein pathways within the liver and the circulation to its advantage.

Differential efficacy of vaccinia virus envelope proteins administered by DNA immunisation in protection of BALB/c mice from a lethal intranasal poxvirus challenge.

DNA vaccines might offer an alternative to the live smallpox vaccine in providing protective efficacy in an orthopoxvirus (OPV) lethal respiratory challenge model. BALB/c mice were immunised with DNA vaccines coding for 10 different single vaccinia virus (VACV) membrane proteins. After an intranasal challenge with the VACV IHD strain, three gene candidates B5R, A33R and A27L produced > or =66% survival. The B5R DNA vaccine consistently produced 100% protection and exhibited greatest efficacy after three 50 microg intramuscular doses in this model. Sero-conversion to these vaccines was often inconsistent, implying that antibody itself was not a correlate of protection. The B5R DNA vaccine induced a strong and consistent gamma interferon (IFNgamma) response in BALB/c mice given a single DNA vaccine dose. Strong IFNgamma responses were also measured in pTB5R immunised C57BL6 mice deficient for MHC class I molecules, suggesting that the memory response was mediated by a CD4+ T cell population.

The use of low molecular weight heparin in pediatric patients: a prospective cohort study.

OBJECTIVE: Low molecular weight heparins (LMWHs) offer several advantages over standard anticoagulant therapy (unfractionated heparin/warfarin) including predictable pharmacokinetics, minimal monitoring, and subcutaneous administration. Our objective was to determine the safety and efficacy of LMWHs in children. METHODS: A prospective cohort of children treated with the LMWH enoxaparin (Rhone Poulenc Rorer) was monitored at the Hospital for Sick Children, Toronto, Canada, from March 1994 until July 1997. RESULTS: There were 146 courses of LMWH administered for treatment and 31 courses for prophylaxis of thromboembolic events (TEs). Clinical resolution of TEs occurred in 94% of children receiving therapeutic doses of LMWH, and 96% of children receiving prophylactic doses of LMWH had no symptoms of recurrent or new TEs. Major bleeding occurred in 5% of children receiving therapeutic doses. Recurrent or new TEs occurred in 1% and 3% of children receiving therapeutic and prophylactic doses of LMWH, respectively. CONCLUSION: LMWH appears to be efficacious and safe for both management and prophylaxis of TEs. The results of this cohort study justify a randomized controlled trial comparing LMWH with standard therapy for the management of TEs in children.

Pharmacokinetics of tolbutamide in ethnic Chinese.

AIMS: Ethnic differences in drug disposition have been described for many drugs. Despite the widespread use of tolbutamide in Asian populations, the pharmacokinetics of tolbutamide, a CYP2C9 substrate, have not been described in ethnic Chinese. METHODS: The pharmacokinetics of tolbutamide (500 mg orally) were studied in 10 young, healthy volunteers (seven male/three female; age 21-29 years), each of whom had four ethnic Chinese grandparents. Plasma concentrations of tolbutamide were measured for 32 h post-dose by high performance liquid chromatography. The concentrations of hydroxytolbutamide and carboxytolbutamide were also measured in urine for 32 h post-dose. Noncompartmental pharmacokinetic parameters were calculated using standard equations and compared with those previously reported in Caucasian subjects using the Mann-Whitney U test. RESULTS: Pharmacokinetic parameters in Chinese (mean+/-s.d.) including Cmax (63+/-11 microg ml(-1)), tmax (median 3.3 h; range 1.6-6.0 h), V/F (9.1+/-1.7 l) and t1/2, (9.1 h; harmonic mean) were similar to the values in Caucasians. CL/F (637+/-88 ml h(-1)) was higher in Chinese than Caucasians. The urinary recoveries of hydroxytolbutamide (13+/-1% of dose) and carboxytolbutamide (68+/-5% of dose) and the partial apparent metabolic clearance (0.15+/-0.02 ml min(-1) kg(-1)) in Chinese were comparable with Caucasians. CONCLUSIONS: The pharmacokinetics of tolbutamide have been described in ethnic Chinese and the disposition is similar to that reported in Caucasians. This study suggests that there is no substantial ethnic difference in the tolbutamide hydroxylase activity of CYP2C9.

Morphological and biochemical features affecting the antithrombotic properties of the aorta in adult rabbits and rabbit pups.

We hypothesised that there are important physiologic differences in arterial wall structure and function with respect to antithrombotic activity in the very young (pre-puberty) compared to adults. Electron microscopy, gel electrophoresis, and activity assays were used to examine differences in aorta structure and function comparing prepubertal rabbits (pups) to adult rabbits. Differences in endothelial function, extracellular matrix structure, proteoglycan (PG) distribution and glycosaminoglycan (GAG) content and function were shown. In both intima and media, total PG, chondroitin sulfate (CS) PG and heparan sulfate (HS) PG content were significantly increased in pups compared to adult rabbits. These findings corresponded to increased concentrations by mass analyses of CS GAG and DS GAG in aortas from pups. There was also a significant increase in antithrombin activity in pups due to HS GAG. In conclusion, differences in both structure and antithrombin activity of aortas from pups compared to adult rabbits suggest that young arteries may have greater antithrombotic potential that is, at least in part, related to increased HS GAG.

Morphologic and biochemical features affecting the antithrombotic properties of the inferior vena cava of rabbit pups and adult rabbits.

The incidence of venous thromboembolic disease is reduced in children compared with adults. Thromboprotective mechanisms, some of which have already been identified in plasma, must be present in children. Blood vessel walls have important antithrombotic properties that maintain blood fluidity. This is in part due to proteoglycan (PG)-related glycosaminoglycan (GAG) molecules within vessel walls. PGs are macromolecules with covalently attached GAG chains, either chondroitin, dermatan, heparan, or keratan sulfate. The influence of age on the concentration and anticoagulant activities of PGs and GAGs, within vein walls before puberty has not been previously investigated. We hypothesized that developmental differences in vein walls may contribute to the reduced risk of thrombosis in children. We used a rabbit model to examine morphologic and biochemical features of inferior venae cavae (IVCs). We assessed IVC wall morphology, PG distribution, GAG mass, and GAG antithrombin activity. Morphologically, there were only minor differences between pups and adult rabbits' IVCs. However, there was a significant increase in GAGs by mass in IVCs from pups compared with adult rabbits (p = 0.012). In addition the total antithrombin activity (p = 0.04), and especially that of heparan sulfate (p = 0.01) was significantly increased in pups compared with adult rabbits. These results demonstrate important differences in the antithrombotic properties of IVC walls in pups and adult rabbits. In summary, developmental differences in vein wall PG content and activity exist which may contribute to the reduced risk of venous thromboembolism in children. Further characterization of these differences is required.

Management of the impacted ureteral calculus.

The management of 42 impacted ureteral calculi is reviewed. Impacted stones were defined by the inability to pass a guide wire or catheter on initial attempts. Stones were impacted in the upper ureter in 10 patients, mid ureter in 11 and lower ureter in 21. Upper ureteral stones were treated in 8 patients by extracorporeal shock wave lithotripsy after disimpaction by laser or other techniques. Mid ureteral stones were treated by laser alone in 7 patients and by extracorporeal shock wave lithotripsy after disimpaction in 4. Lower stones were treated by laser in 17 patients and ultrasound in 2. Complications included 3 major and 5 minor perforations, and 4 false passages. Treatment was successful without an open operation in 40 of 42 patients (95%). Our current approach to impacted ureteral calculi involves passing a rigid ureteroscope to the stone, with disimpaction performed by laser fragmentation or other dislodgement maneuvers. Proximal stones or large fragments then are treated by extracorporeal shock wave lithotripsy. Mid ureteral stones are treated similarly, unless they are so fragile that in situ fragmentation may be completed easily. Lower ureteral stones are fragmented in situ, with hard fragments extracted by basket. Alternative treatments for impacted calculi at all levels include unstented in situ extracorporeal shock wave lithotripsy, antegrade ureteroscopy and, finally, an operation.

The effects of various indwelling ureteral catheter materials on the normal canine ureter.

In an attempt to objectively evaluate the biocompatibility of materials commonly used for ureteral stenting, stent-catheters made of four different materials were placed randomly in 31 ureters of 19 mongrel dogs. Animals underwent urography at four weeks and were sacrificed at six weeks. Mild hydronephrosis was noted in eight instances, essentially unrelated to specific material. Silicone, C-flex and polyurethane stents caused a similar, mild degree of ureteral edema, but ureters stented with Silitek demonstrated fairly marked edema. Epithelial ulceration and erosion, often severe, occurred with all polyurethane stents, and rarely with the three other materials. All materials differed statistically from controls, and C-flex caused less reaction overall than polyurethane, indicating differences in biocompatibility of the various materials which may be relevant to their use in patients undergoing long-term ureteral stenting. In practical terms, these suggest that certain materials, notably silicone and C-flex, are more suitable for ureteral stenting than others.

The dexamethasone suppression test: importance of dexamethasone concentrations.

Plasma dexamethasone concentrations and cortisol response to dexamethasone were measured in 29 normal healthy volunteers, 23 depressed patients, and 10 patients with anorexia nervosa at 4:00 PM postdexamethasone. In each of the 3 groups, nonsuppressors had lower dexamethasone concentrations than suppressors. Of the subjects with plasma dexamethasone at or below 0.7 ng/ml, a significantly higher proportion (48%) were nonsuppressors compared to the proportion above 0.7 ng/ml (14%), all of whom were patients. Plasma dexamethasone concentrations in a subgroup of depressed nonsuppressors were high (mean 1.35 ng/ml), whereas the remainder were low (0.42 ng/ml) and were similar to the normal nonsuppressors (0.35 ng/ml), suggesting different mechanisms for nonsuppression in the subgroups. Plasma dexamethasone concentrations were similar in nonendogenous and endogenous depressives, in men and women, and in medicated and drug-free patients. None of the variables of age, weight, history of weight loss, Hamilton depression rating score, predexamethasone cortisol, or postdexamethasone cortisol were significantly correlated with plasma dexamethasone, except for body weight and a history of weight loss in the depressed group only. Mean plasma dexamethasone concentrations increased significantly from week 1 to week 2 in 7 depressed patients, whereas plasma cortisol decreased; however, the relationship between dexamethasone and cortisol varied considerably for individual patients.

No change in neostriatal D-2 dopamine receptors after NMDA lesions of rat prefrontal cortex.

D-2 dopamine receptor function in rat nucleus accumbens and anterior corpus striatum was examined 6-8 days following N-methyl-D-aspartate lesions confined to medial prefrontal cortex. Lesions failed to alter the affinity or density of D-2 receptors labelled by [3H]spiperone in membrane preparations of both subcortical areas. Locomotor activity and stereotyped behaviours induced by the D-2 agonist, LY-171555, were also not significantly altered in lesioned animals. These results suggest that D-2 dopamine receptors of nucleus accumbens and anterior corpus striatum are not localized on corticofugal afferents from medial prefrontal cortex.

Excitotoxin lesions suggest an aspartatergic projection from rat medial prefrontal cortex to ventral tegmental area.

High affinity D-[3H]aspartate uptake and amino acid concentrations were examined in synaptosome-enriched preparations of microdissected rat ventral tegmental area 6-7 days following N-methyl-D-aspartate lesions confined to medial prefrontal cortex. Specific reductions were observed in the high affinity uptake of D-[3H]aspartate (59% of control, P less than 0.005) and concentrations of L-aspartate (79% of control, P less than 0.05) in the ventral tegmental area suggesting the presence of an aspartatergic projection from medial prefrontal cortex to this area.

Applicability of the Colebrook-White Formula to Represent Frictional Losses in Partially Filled Unsteady Pipeflow.

The use of Manning's n as a friction factor is shown to be unsuitable in the case of small bore (less than about one meter diameter) partially filled pipeflow, particularly for relatively smooth materials such as glass and cast-iron. The Colebrook-White equation with the roughness coefficient k is presented in a form suitable for inclusion in a computer program to solve the partially filled unsteady pipeflow equations by means of the method of characteristics. Results are presented which show that the Colebrook-White equation provides substantially improved predictions of the wave velocity along the pipe. It provides slightly improved predictions for the maximum depth of flow along the pipe.

75Se-release: a short and long term assay system for cellular cytoxicity.

The gamma-emitting aminoacid 75Se-selenomethionine (75SeM) was examined as a target cell label in cytotoxic assays. It was efficiently taken up by activated, intensively metabolizing cells of various types but hardly at all by resting or low-metabolizing cells. Culturing activated cells in methionine-deficient medium with 3--5 mu Ci75SeM/ml for 18--22 h usually resulted in an uptake of 3--20 cpm/cell which was 3--200 times that of 51Cr marked cells. 75SeM-labelled cells kept in medium at ambient temperature or at 37 degrees C, maintained a high radioactivity per cell and a viability above 85% for at least 72 h without significant increase in spontaneous isotope release or loss in sensitivity in subsequent cytotoxic tests. 75Se-labelled material released from target cells was not reutilized by unlabelled lymphoid cells. Provided the cells were carefully washed after labelling and kept in optimal culture conditions, the reasonably low baseline release (usually 0.6--1.8% of input/h) in the medium control allowed performance of long-term assays of up to 54 h. However, strong cytotoxic reactions (e.g. ADCC) could cause over 50% specific 75Se-release within 5 h. With constant amounts of effector cells (3.6 x 10(3) up to 3 x 10(5)/well) identical or even higher, specific releases were obtained on 6 x 10(2) targets as compared to 1 x 10(4) targets/well. Thus, the 75Se-release assay offers a single monitoring system suitable for short (3--6 h) and long term (usually up to 44 h) cytotoxic reactions on a microscale, using 1 x 10(3) or less targets/well. Its sensitivity permits evaluation of strong and weak reactions as well as early and delayed onset cytotoxicity. In addition, with a gamma-spectrometer the radioactivity of 75Se can easily be distinguished from that of 51Cr. Due to this, and an improved method for 51Cr labelling of cells (10 mu Ci 51Cr/ml medium for 18--22 h), a double gamma-labelling of cellular proteins is available which provides new possibilities for monitoring cellular interactions in short and long term tests.