Mitochondrial J haplogroup is associated with lower blood pressure and anti-oxidant status: findings in octo/nonagenarians from the BELFAST Study

Mitochondria produce cellular energy but also free-radicals, which damage cells despite an array of endogenous anti-oxidants. In Northern Europe, the mitochondrial haplogroup J has been related to longevity in nonagenarians and centenarians but also with age-related disease. Hypertension is an important contributor to atherosclerotic-related diseases and its pathogenesis is associated with increased oxidative stress. In this study, we questioned whether J haplogroup octo/nonagenarians from the Belfast Elderly Longitudinal Free-living Elderly STudy (BELFAST) study showed evidence of protective blood pressure or anti-oxidant profile which might explain their longevity advantage. Briefly, in a cross-sectional study, community-living, mentally alert (Folstein >25/30), octo/nonagenarian subjects, recruited for good health, were enlisted and consented as part of the BELFAST study, for blood pressure, anthropometric measurements and blood sampling. DNA typing for mitochondrial haplotypes was carried out with measurements for enzymatic and non-enzymatic antioxidants. J haplogroup carriers showed lower systolic blood pressure and glutathione peroxidase activity (Gpx) with higher folate measurements. There was no change in urate, bilirubin, albumin or nutrition-related antioxidants-selenium or vitamins A, C and a and ß carotene. BELFAST study mtDNA J haplogroup octo/nonagenarians showed lower blood pressure and reduced glutathione peroxidase activity and higher folate, but no change for other antioxidants. These findings are of interest in view of mtDNA J haplogroup's association with increased age in some previous studies.

Response of red blood cell folate to intervention: implications for folate recommendations for the prevention of neural tube defects

Committees worldwide have set almost identical folate recommendations for the prevention of the first occurrence of neural tube defects (NTDs). We evaluate these recommendations by reviewing the results of intervention studies that examined the response of red blood cell folate to altered folate intake. Three options are suggested to achieve the extra 400 mu g folic acid/d being recommended by the official committees: increased intake of folate-rich foods, dietary folic acid supplementation, and folic acid fortification of food. A significant increase in foods naturally rich in folates was shown to be a relatively ineffective means of increasing red blood cell folate status in women compared with equivalent intakes of folic acid-fortified food, presumably because the synthetic form of the vitamin is more stable and more bioavailable. Although folic acid supplements are highly effective in optimizing folate status, supplementation is not an effective strategy for the primary prevention of NTDs because of poor compliance. Thus, food fortification is seen by many as the only option likely to succeed. Mandatory folic acid fortification of grain products was introduced recently in the United States at a level projected to provide an additional mean intake of 100 mu g folic acid/d, but some feel that this policy does not go far enough. A recent clinical trial predicted that the additional intake of folic acid in the United States will reduce NTDs by >20%, whereas 200 mu g/d would be highly protective and is the dose also shown to be optimal in lowering plasma homocysteine, with possible benefits in preventing cardiovascular disease. Thus, an amount lower than the current target of an extra 400 mu g/d may be sufficient to increase red blood cell folate to concentrations associated with the lowest risk of NTDs, but further investigation is warranted to establish the optimal amount.

Comparison of serum and whole-blood specimens for the detection of Candida DNA in critically ill, non-neutropenic patients

In contrast to the multitude of studies on fungal PCR assay methods, little work has been reported evaluating Candida PCR performance when using whole blood compared with serum in candidaemic patients. Here, a comparison of the performance of whole-blood and serum specimens using a set of real-time PCR Candida species assays is described. Specimens were collected prospectively from non-neutropenic adults who were recruited to a diagnostic clinical trial, the primary purpose of which was to verify the performance of the assays using serum; in all, 104 participants also had whole-blood specimens submitted for analysis in addition to the serum specimen. Of these participants, 10 had laboratory-confirmed candidaemia and 94 were categorized as being 'unlikely' to have invasive Candida infection. PCR results from the whole-blood specimens are presented here and compared with the results from serum specimens in this subgroup among whom both specimen types were obtained contemporaneously. All participants with candidaemia were PCR-positive from serum samples; however, only seven were PCR-positive from whole blood. All specimens from patients in the 'unlikely' category were PCR-negative in both types of specimen. Moreover, DNA extraction from serum required 1 h; extraction from whole blood required approximately 3 h. These data tentatively suggest that, overall, serum is an appropriate specimen for Candida PCR for detection of candidaemia in non-neutropenic adults.

Improving molecular detection of Candida DNA in whole blood:comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 1 8S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100% concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from ~72 to

Comparison of media for optimal recovery of Candida albicans and Candida glabrata from blood culture

Candida spp., mainly Candida albicans, are frequently responsible for complications in immunocompromised patients. There are limited data comparing recovery efficiency using simple non-selective basal broth media.

Impact of Age on the Importance of Systolic and Diastolic Blood Pressures for Stroke Risk:The MOnica, Risk, Genetics, Archiving, and Monograph (MORGAM) Project

This study investigates age-related shifts in the relative importance of systolic (SBP) and diastolic (DBP) blood pressures as predictors of stroke and whether these relations are influenced by other cardiovascular risk factors. Using 34 European cohorts from the MOnica, Risk, Genetics, Archiving, and Monograph (MORGAM) Project with baseline between 1982 and 1997, 68 551 subjects aged 19 to 78 years, without cardiovascular disease and not receiving antihypertensive treatment, were included. During a mean of 13.2 years of follow-up, stroke incidence was 2.8%. Stroke risk was analyzed using hazard ratios per 10-mm Hg/5-mm Hg increase in SBP/DBP by multivariate-adjusted Cox regressions, including SBP and DBP simultaneously. Because of nonlinearity, DBP was analyzed separately for DBP =71 mm Hg and DBP

Age-related reference intervals for lymphocyte subsets in whole blood of healthy individuals

Enumeration of various lymphocyte subsets is used widely in the diagnosis and monitoring of various disease states. With the development of flow cytometric technology and whole blood analysis, methodologies have become more sensitive. It is therefore important to establish reference intervals in normal, healthy individuals using these techniques to give a better indication of the border between health and disease. Since some lymphocyte subpopulations are known to change with age, we have enumerated common subsets in healthy individuals from all decades of adult life, including nonagenarian subjects. We report reference intervals for these subsets in each age group, which will be of use in diagnosis and disease monitoring, particularly in elderly subjects, the most rapidly expanding group within the population today.

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE(-/-) mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE(-/-) mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.