[What makes a parasite "transforming"? Insights into cancer from the agents of an exotic pathology, Theileria spp]. / Comment et pourquoi un parasite peut-il être « transformant ¼ ? Apports d'agents de zoonoses exotiques, Theileria spp., à l'étude du cancer.

Theileria are obligate eukaryotic intracellular parasites of cattle. The diseases they cause, Tropical theileriosis and East Coast Fever, cause huge economic loss in East African, Mediterranean and central and South-East Asian countries. These apicomplexan parasites are the only intracellular eukaryotic parasites known to transform their host cell and represent a unique model to study host-parasite interactions and mechanisms of cancer onset.Here, we review how Theileria parasites induce transformation of their leukocyte host cell and discuss similarities with tumorigenesis. We describe how genomic innovation, epigenetic changes and hijacking of signal transductions enable a eukaryotic parasite to transform its host cell.

Evaluation of the bactericidal efficacy of three different alcohol hand rubs against clinical isolates of Staphylococcus aureus using an ex vivo carrier test.

We tested the efficacy of three alcohol hand rubs (AHRs) against Staphylococcus aureus using an ex vivo carrier test method and investigated the residual activity of AHRs and the effect of mechanical rubbing. A much longer contact time was required for the AHRs to achieve a bactericidal effect using the ex vivo test (between 10 and >20 min) compared with the in vitro test. Mechanical rubbing was found to increase the efficacy of the AHR compared to a rubbing control. Since the AHRs had no residual activity, the bactericidal effect achieved using the ex vivo test with contact times greater than the evaporation times (15 s) is unlikely to be achieved in practice. In view of such findings it is unlikely that AHRs are able to achieve a significant bactericidal effect (≥4 log(10) reduction) in practice, suggesting that contamination on the hands of healthcare workers (HCWs) may not be reduced enough to overcome the risk of cross-contamination and healthcare-associated infection. Since the AHRs had no residual effect they would be unable to prevent recolonisation of the hands of HCWs.

Evaluation of the bactericidal efficacy of three different alcohol hand rubs against 57 clinical isolates of S. aureus.

We tested the efficacy of three alcohol hand rubs (AHRs) used in two local Welsh intensive therapy units (ITUs) against Staphylococcus aureus. The test protocol was based on a carrier test and parameters (concentration, contact time) were chosen following observation of hand-sanitising practices in the ITUs. Following AHR exposure, surviving bacteria were enumerated using a standard plate count method plus a Bioscreen C Microbial Growth Analyser. The AHRs demonstrated variable efficacy against the clinical isolates: the mean log(10) reduction after 10 s exposure to Soft Care Med H5, Cutan and Guest Medical AHRs was 2.67, 0.696 and 1.96, respectively, and after 30 s exposure was 4.58, 1.74 and 3.60, respectively. Since the average time taken by healthcare workers (HCWs) to rub AHR onto their hands was 11 s and 15 s at the two hospitals, the efficacy of these AHRs may be significantly limited against the S. aureus isolates under the conditions observed in practice. In addition, differences observed in log(10) reduction in bacterial number post-exposure using the Bioscreen compared to the plate count method provided evidence that S. aureus may be able to recover following Guest Medical AHR treatment within 2 min exposure, whereas after 5 min exposure bacterial damage caused by the AHR was irreversible. Although the introduction of AHRs improved hand hygiene compliance among HCWs, our observations highlighted that contact time is an important factor to ensure the efficacy of these products.

Pharmacogenetics--legal, ethical and regulatory considerations.

The overall objective of pharmacogenetics is to determine the genetic basis of variability in drug efficacy and safety, and to use this information to benefit the patient. Genetics can be used to develop drugs that are suitable for the majority of patients and to help identify those patients for whom a certain drug may not be the most appropriate. This review will cover some background to pharmacogenetics and various issues including confidentiality, data protection, coding of samples and genetic data, informed consent, and drug development guidelines. International, national and regional variation in the legal and regulatory basis for pharmacogenetics presents challenges for researchers attempting to increase scientific understanding in the field. Examples of national and international regulations and guidelines will be given. It is clear that pharmacogenetics today is a long way from the 'personalised medicine' advocated by some individuals in recent years. The aim of pharmacogenetic research should always be to make sure that patients have the best treatment available and that patients are not exposed to drugs to which they are genetically unable to respond. This vision must continue to inspire researchers and regulators who are working together to make it a widespread reality.

Vitamin E protects against iron-hexachlorobenzene induced porphyria and formation of 8-hydroxydeoxyguanosine in the liver of C57BL/10ScSn mice.

The effect of vitamin E treatment on total porphyrin content, lipid peroxidation (LOOH) and 8-hydroxydeoxyguanosine (8-OHdG) was studied in the livers of C57BL/10ScSn mice following hexachlorobenzene (HCB) and iron treatment. HCB was administered i.p. (totalling 300 mg/kg) twice, with 1 week interval. Three days after the first HCB injection iron-dextran was given i.p. (500 mg Fe per kg). Vitamin E was administered weekly (20 mg/kg) by s.c. injection. Both total hepatic porphyrin and LOOH levels were significantly (P<0.001) increased in the HCB-iron treated group as compared with the control group. Mice treated additionally with vitamin E had significant (P<0.001) lower levels as compared with the HCB-iron group. Similarly, the levels of 8-OHdG were significantly (P<0.001) increased above controls after HCB-iron treatment and this increase was reduced after co-treatment with vitamin E (P<0.02). The data support the hypothesis that the mechanism of hepatic porphyrinogenicity of HCB with iron overload is an oxidative free radical process.

The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture.

The CHO320 cell line, engineered to produce human interferon gamma was investigated with regard to its susceptibility to oxidative stress. Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20%. High concentrations of hydrogen peroxide (in excess of 200 microM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 microns and 100 microM had no effect on recombinant protein production. Buthionine sulphoximine (50 microM and 100 microM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen. It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress.

Anger suppression, reactivity, and hypertension risk: gender makes a difference.

The present study investigated gender-related differences in cardiovascular reactivity and the role of anger inhibition and risk for future hypertension. Tonic blood pressure served as an index of hypertension risk. Twenty-eight female and 26 male college students with high and low normal blood pressure were recruited on the basis of their mean arterial pressure. Continuous measures of heart rate and blood pressure were taken while participants carried out a series of behavioral manoeuvres including mental arithmetic, interpersonal challenge, a frustrating psychomotor test, and the cold pressor test. Participants also completed inventories assessing trait anxiety, trait anger, anger expression, and Type A. The results are in concordance with previous findings and show higher cardiovascular reactivity in men than in women and in subjects at risk for hypertension. Within the male group, a combination of hypertension risk and anger suppression led to the highest reactivity, whereas in female subjects, differences in anger-in had no effect on reactivity. The implications of these results are discussed in light of sex differences in cardiovascular morbidity and mortality.

Susceptibility of gamma-irradiated proteins to in vitro glycation: exposure to oxygen free radicals increases glycation-induced modifications.

Oxidation and glycation are non-enzymatic protein modifications involved in the pathogenesis of aging. We evaluated their possible influences in an in vitro system: albumin was oxidized by gamma-irradiation and then exposed to glycation in vitro. Fluorescence modifications were analysed as signals of protein alterations. Both radiolytic oxidation and in vitro glycation provoked a sharp decrease of tryptophan fluorescence (278 nm ex./340 nm em.); their effects tended to be additive, unless a saturation limit was reached. Both individually and in combination, these two non-enzymatic processes induced the appearance of a new fluorescence (335 nm ex./415 nm em.); in this case as well there was an additive effect, with a trend toward saturation. Radiolytic oxidation and in vitro glycation seem to provoke similar damage to the exposed proteins: the observed fluorescence alterations may be due to similar conformational changes, breaks or the development of fluorophores.

Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS)

BACKGROUND: Vitamin E (alpha-tocopherol) is thought to have a role in prevention of atherosclerosis, through inhibition of oxidation of low-density lipoprotein. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of alpha-tocopherol and lower rates of ischaemic heart disease. We tested the hypothesis that treatment with a high dose of alpha-tocopherol would reduce subsequent risk of myocardial infarction (MI) and cardiovascular death in patients with established ischaemic heart disease. METHODS: In this double-blind, placebo-controlled study with stratified randomisation, 2002 patients with angiographically proven coronary atherosclerosis were enrolled and followed up for a median of 510 days (range 3-981). 1035 patients were assigned alpha-tocopherol (capsules containing 800 IU daily for first 546 patients; 400 IU daily for remainder); 967 received identical placebo capsules. The primary endpoints were a combination of cardiovascular death and non-fatal MI as well as non-fatal MI alone. FINDINGS: Plasma alpha-tocopherol concentrations (measured in subsets of patients) rose in the actively treated group (from baseline mean 34.2 micromol/L to 51.1 micromol/L with 400 IU daily and 64.5 micromol/L with 800 IU daily) but did not change in the placebo group. Alpha-tocopherol treatment significantly reduced the risk of the primary trial endpoint of cardiovascular death and non-fatal MI (41 vs 64 events; relative risk 0.53 [95% Cl 0.34-0.83; p=0.005). The beneficial effects on this composite endpoint were due to a significant reduction in the risk of non-fatal MI (14 vs 41; 0.23 [0.11-0.47]; p=0.005); however, there was a non-significant excess of cardiovascular deaths in the alpha-tocopherol group (27 vs 23; 1.18 [0.62-2.27]; p=0.61). All-cause mortality was 36 of 1035 alpha-tocopherol-treated patients and 27 of 967 placebo recipients. INTERPRETATION: We conclude that in patients with angiographically proven symptomatic coronary atherosclerosis, alpha-tocopherol treatment substantially reduces the rate of non-fatal MI, with beneficial effects apparent after 1 year of treatment. The effect of alpha-tocopherol treatment on cardiovascular deaths requires further study.

Equally efficacious asthma management with budesonide 800 micrograms administered by Turbuhaler or with beclomethasone dipropionate > or = 1500 micrograms given through a pressurized metered-dose inhaler with spacer. The French Budesonide Trial Group.

To avoid the side effects associated with long-term administration of high doses of inhaled glucocorticosteroids, they should be used at the lowest effective dose. This study compared the clinical efficacy of budesonide given via a dry-powder, inspiratory flow-driven device (Turbuhaler), at a daily dose of 800 micrograms, with beclomethasone dipropionate (BDP) 1500 to 2000 micrograms given via pressurized metered-dose inhaler (pMDI) with spacer to adults requiring the latter dose of BDP to control their asthma. The study was performed as a 2-week run-in, 8-week open, randomized, multicenter, parallel-group design. Adult asthmatics with a forced expiratory volume in 1 second 55% or more of predicted normal and receiving BDP 1500 to 2000 micrograms daily entered the study. After a 2-week run-in, one group continued with BDP and the other was switched to budesonide through the Turbuhaler. After 8 weeks, morning peak expiratory flow (PEF) had increased by 5.9 L/min from a mean of 390 L/min in the budesonide group and by 1.9 L/min from a mean of 402 L/min in the BDP group. No clinically or statistically significant differences between groups were evident with regard to the change in this primary variable. Similarly, only small changes in evening PEF and secondary variables of lung function were seen, with no statistically significant difference between groups. The authors concluded that both treatments were equivalent in managing asthma in adult patients with stable asthma.

Free radical reactions involving the angiotensin converting enzyme inhibitor captopril.

Using the pulse radiolysis technique, absolute rate constants have been obtained for the reaction of captopril with several free radicals. The results demonstrate that although captopril reacts rapidly with a number of free radicals, such as the hydroxyl radical (k = 5.1 × 10(9) dm(-3)mol(-1)s(-1)) and the thiocyanate radical anion (k = 1.3 × 10(7) dm(-3)mol(-1)s(-1)), it is not exceptional in this ability. Similarly, the reactions with carbon centred radicals although rapid are an order of magnitude slower than those observed with glutathione. Additional lipid peroxidation studies further demonstrate that captopril is a much less effective antioxidant than glutathione. The data go some way to supporting the view that any attenuation of reperfusion injury by captopril is not through a direct free radical scavenging mechanism but may be afforded by other, non-radical-mediated mechanisms.

Depletion of alpha-tocopherol in human atherosclerotic lesions.

Estimations of alpha-tocopherol content were made on a series of human necropsy samples of normal arterial wall and of atherosclerotic lesions. The results were compared with stage of lesion, shown by histology, and with the amounts of cholesterol and hydroxycholesterols in the same lesions. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal arterial wall but was consistently low in lesions, especially in lesions rich in macrophage foam cells. The results suggested that significant accumulation of hydroxycholesterols, found almost exclusively in lesions, only occurred when alpha-tocopherol levels were low in relation to the cholesterol content. This suggests that oxidative activity in the lesion may lead to significant oxidation of constituents of low-density lipoprotein only after alpha-tocopherol has been depleted.

Biokinetics in humans of RRR-alpha-tocopherol: the free phenol, acetate ester, and succinate ester forms of vitamin E.

The bioavailability of RRR-alpha-tocopherol from the oral administration of RRR-alpha-tocopherol itself and its acetate and succinate esters was determined in healthy human subjects. Venous blood samples were withdrawn periodically over a 51-h period following oral administration of a gelatin capsule containing an equimolar mixture of RRR-alpha-tocopherol and RRR-alpha-tocopheryl acetate. In a second study, subjects received a capsule containing an equimolar mixture of RRR-alpha-tocopheryl acetate and RRR-alpha-tocopheryl succinate. In Study 1, RRR-alpha-tocopherol was absorbed at similar rates from both the free phenol, and the acetate ester and maximum plasma levels occurred at 12 h in most subjects. The extent of absorption of RRR-alpha-tocopherol varied considerably between subjects in absolute terms, but the relative absorption from the two forms was remarkably consistent, and a ratio of 1.0 was found for parameters of relative bioavailability in plasma. The concentration of RRR-alpha-tocopherol from each form was maximal at approximately 27 h in red blood cells and, as seen with the plasma data, there was a large interindividual variability. In Study 2, there was no significant difference in the extent of absorption of RRR-alpha-tocopherol from the acetate ester and the succinate ester, although there was an apparently higher initial rate of absorption from the acetate ester.

Exposure of beta H-crystallin to hydroxyl radicals enhances the transglutaminase-susceptibility of its existing amine-donor and amine-acceptor sites.

beta H-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations, and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [14C]Methylamine was used as a probe for labelling amine-acceptor sites; a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established, and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be beta B1-, beta A3- and beta A4-crystallin. These data support the proposal that oxidative stress and transglutaminase activity may be jointly involved in the changes found in lens crystallins with age and in the development of cataract.

Measurement of n-alkanals and hydroxyalkenals in biological samples.

A modified method was developed to measure nM levels of a range of n-alkanals and hydroxyalkenals in biological samples such as blood plasma and tissue homogenates and also in Folch lipid extracts of these samples. Butylated hydroxytoluene (BHT) and desferrioxamine (Desferal) were added to samples to prevent artifactual peroxidation. Aldehydes were reacted with 1,3-cyclohexanedione (CHD), cleaned up by solid-phase extraction on a Sep-Pak C18 cartridge and the fluorescent decahydroacridine derivatives resolved by reverse-phase high-performance liquid chromatography (HPLC) with gradient elution. A wider range of aldehydes was detected in lipid extracts of plasma and liver homogenate compared to whole (unextracted) samples. Human plasma contained nM levels of acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal. 4-Hydroxynonenal (0.93 nmol/g) and alkanals with two to six carbons (up to 7.36 nmol/g) were detected in rat liver. Recovery of aldehydes added to whole plasma or to lipid extracts of plasma was dependent on carbon chain length, varying from 95% for acetaldehyde to 8% for decanal. Recovery from biological samples was significantly less than that of standards taken through the Sep-Pak clean-up procedure, suggesting that aldehydes can bind to plasma protein and lipid components.

An introduction to free radical biochemistry.

Free radicals are chemical species possessing an unpaired electron that can be considered as fragments of molecules and which are generally very reactive. They are produced continuously in cells either as accidental by-products of metabolism or deliberately during, for example, phagocytosis. The most important reactants in free radical biochemistry in aerobic cells are oxygen and its radical derivatives (superoxide and hydroxyl radical), hydrogen peroxide and transition metals. Cells have developed a comprehensive array of antioxidant defences to prevent free radical formation or limit their damaging effects. These include enzymes to decompose peroxides, proteins to sequester transition metals and a range of compounds to 'scavenge' free radicals. Reactive free radicals formed within cells can oxidise biomolecules and lead to cell death and tissue injury. Establishing the involvement of free radicals in the pathogenesis of a disease is extremely difficult due to the short lifetimes of these species.