Low-dose vitamin B-6 effectively lowers fasting plasma homocysteine in healthy elderly persons who are folate and riboflavin replete.

BACKGROUND: Current data suggest that physiologic doses of vitamin B-6 have no significant homocysteine-lowering effect. It is possible that an effect of vitamin B-6 was missed in previous trials because of a much greater effect of folic acid, vitamin B-12, or both. OBJECTIVE: The aim of this study was to investigate the effect of low-dose vitamin B-6 supplementation on fasting total homocysteine (tHcy) concentrations in healthy elderly persons who were made replete with folate and riboflavin. DESIGN: Twenty-two healthy elderly persons aged 63-80 y were supplemented with a low dose of vitamin B-6 (1.6 mg/d) for 12 wk in a randomized, double-blind, placebo-controlled trial after repletion with folic acid (400 microg/d for 6 wk) and riboflavin (1.6 mg/d for 18 wk); none of the subjects had a vitamin B-12 deficiency. RESULTS: Folic acid supplementation lowered fasting tHcy by 19.6% (P

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOS298Asp revealed a 135-kDa proteolytic fragment which was not present with eNOS298Glu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu298 and Asp298 eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.

A randomised interventional trial of ω-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus

Objective: To determine the clinical effect of dietary supplementation with low-dose ?-3-polyunsaturated fatty acids on disease activity and endothelial function in patients with systemic lupus erythematosus. Methods: A 24-week randomised double-blind placebo-controlled parallel trial of the effect of 3 g of ?-3-polyunsaturated fatty acids on 60 patients with systemic lupus erythematosus was performed. Serial measurements of disease activity using the revised Systemic Lupus Activity Measure (SLAM-R) and British Isles Lupus Assessment Group index of disease activity for systemic lupus erythematosus (BILAG), endothelial function using flow-mediated dilation (FMD) of the brachial artery, oxidative stress using platelet 8-isoprostanes and analysis of platelet membrane fatty acids were taken at baseline, 12 and 24 weeks. Results: In the fish oil group there was a significant improvement at 24 weeks in SLAM-R (from 9.4 (SD 3.0) to 6.3 (2.5), p

Advanced glycation of fibronectin impairs vascular repair by endothelial progenitor cells: Implications for vasodegeneration in diabetic retinopathy

PURPOSE. Vascular repair by marrow-derived endothelial progenitor cells (EPCs) is impaired during diabetes, although the precise mechanism of this dysfunction remains unknown. The hypothesis for the study was that progressive basement membrane (BM) modification by advanced glycation end products (AGEs) contributes to impairment of EPC reparative function after diabetes-related endothelial injury.

METHODS. EPCs isolated from peripheral blood were characterized by immunocytochemistry and flow cytometry. EPC interactions on native or AGE-modified fibronectin (AGE-FN) were studied for attachment and spreading, whereas chemotaxis to SDF-1 was assessed with the Dunn chamber assay. In addition, photoreactive agent-treated monolayers of retinal microvascular endothelial cells (RMECs) produced circumscribed areas of apoptosis and the ability of EPCs to “endothelialize” these wounds was evaluated.

RESULTS. EPC attachment and spreading on AGE-FN was reduced compared with control cells (P < 0.05–0.01) but was significantly restored by pretreatment with Arg-Gly-Asp (RGD). Chemotaxis of EPCs was abolished on AGE-FN but was reversed by treatment with exogenous RGD. On wounded RMEC monolayers, EPCs showed clustering at the wound site, compared with untreated regions (P < 0.001); AGE-FN significantly reduced this targeting response (P < 0.05). RGD supplementation enhanced EPC incorporation in the monolayer, as determined by EPC participation in tight junction formation and restoration of transendothelial electric resistance (TEER).

CONCLUSIONS. AGE-modification of vascular substrates impairs EPC adhesion, spreading, and migration; and alteration of the RGD integrin recognition motif plays a key role in these responses. The presence of AGE adducts on BM compromises repair by EPC with implications for vasodegeneration during diabetic microvasculopathy.

Vitamin D deficiency and insufficiency in pregnant women: a longitudinal study.

Maternal vitamin D insufficiency is associated with childhood rickets and longer-term problems including schizophrenia and type 1 diabetes. Whilst maternal vitamin D insufficiency is common in mothers with highly pigmented skin, little is known about vitamin D status of Caucasian pregnant women. The aim was to investigate vitamin D status in healthy Caucasian pregnant women and a group of age-matched non-pregnant controls living at 54–55°N. In a longitudinal study, plasma 25-hydroxyvitamin D (25(OH)D) was assessed in ninety-nine pregnant women at 12, 20 and 35 weeks of gestation, and in thirty-eight non-pregnant women sampled concurrently. Plasma 25(OH)D concentrations were lower in pregnant women compared to non-pregnant women (P < 0·0001). Of the pregnant women, 35, 44 and 16 % were classified as vitamin D deficient (25(OH)D < 25 nmol/l), and 96, 96 and 75 % were classified as vitamin D insufficient (25(OH)D < 50 nmol/l) at 12, 20 and 35 weeks gestation, respectively. Vitamin D status was higher in pregnant women who reported taking multivitamin supplements at 12 (P < 0·0001), 20 (P = 0·001) and 35 (P = 0·001) weeks gestation than in non-supplement users. Vitamin D insufficiency is evident in pregnant women living at 54–55°N. Women reporting use of vitamin D-containing supplements had higher vitamin D status, however, vitamin D insufficiency was still evident even in the face of supplement use. Given the potential consequences of hypovitaminosis D on health outcomes, vitamin D supplementation, perhaps at higher doses than currently available, is needed to improve maternal vitamin D nutriture.

In Vivo Age Dependency of Unfractionated Heparin in Infants and Children

Introduction
Unfractionated Heparin (UFH) is used widely in paediatrics. Paediatric specific recommendations for UFH therapy are few, with the majority of recommendations being extrapolated from adult practice. In vitro studies have shown that this practice may be suboptimal. This study aimed to improve the understanding of the impact of age upon UFH response in vivo.

Materials and Methods
This prospective, observational study, conducted in the Paediatric Intensive Care Unit (PICU), included: patients 16 years or younger; treated with UFH of at least 10 U/Kg/hr. Laboratory analysis included: Antithrombin, APTT, Anti-Xa, Anti-IIa and thrombin generation expressed as the Endogenous Thrombin Potential. Results were grouped according to patient age (i.e. < 1, 1-5, 6-10 and 11-16 years).

Results
85 patients received an equivalent mean UFH dose with a median duration of 3 days. Antithrombin levels were decreased compared to age-related norms in children up to 11 years of age. APTT results were comparable across the age-groups. The Anti-Xa results using two different assays showed a trend for lower values in younger children. All children less than one year old recorded Anti-Xa values outside the therapeutic range for heparin therapy, for both assays. There was a trend for decreased Anti-IIa activity in younger children. Endogenous Thrombin Potential showed a significant trend for increased inhibition in older children. In vitro Antithrombin supplementation did not change the Anti-Xa or thrombin generation.

Conclusions
This study confirms that, in vivo, for the same dose of UFH, the anti Xa and anti IIa effect, as well as the inhibition of endogenous thrombin potential is age dependent and that these differences are not purely AT dependent. The implication is that the anticoagulant and antithrombotic effect of a given dose of UFH differs with age. Clinical outcome studies to determine the optimal dosing for each age group are warranted.

Abbreviations
UFH, Unfractionated Heparin; ETP, Endogenous Thrombin Potential; AT, Antithrombin; APTT, Activated Partial Thromboplastin Time

Molecular detection of exercise-induced free radicals following ascorbate prophylaxis in type 1 diabetes mellitus: a randomised controlled trial

Aims/hypothesis: Patients with type 1 diabetes mellitus are more susceptible than healthy individuals to exercise-induced oxidative stress and vascular endothelial dysfunction, which has important implications for the progression of disease. Thus, in the present study, we designed a randomised double-blind, placebo-controlled trial to test the original hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus. Methods: All data were collected from hospitalised diabetic patients. The electron paramagnetic resonance spectroscopic detection of spin-trapped a-phenyl-tert-butylnitrone (PBN) adducts was combined with the use of supporting markers of lipid peroxidation and non-enzymatic antioxidants to assess exercise-induced oxidative stress in male patients with type 1 diabetes (HbA1c 7.9±1%, n=12) and healthy controls (HbA1c 4.6±0.5%, n=14). Following participant randomisation using numbers in a sealed envelope, venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2 h after oral ingestion of 1 g ascorbate or placebo. Participants and lead investigators were blinded to the administration of either placebo or ascorbate treatments. Primary outcome was the difference in changes in free radicals following ascorbate ingestion. Resuts: Six diabetic patients and seven healthy control participants were randomised to each of the placebo and ascorbate groups. Diabetic patients (n=12) exhibited an elevated concentration of PBN adducts (p<0.05 vs healthy, n=14), which were confirmed as secondary, lipid-derived oxygen-centred alkoxyl (RO•) radicals (a nitrogen=1.37 mT and aßhydrogen=0.18 mT). Lipid hydroperoxides were also selectively elevated and associated with a depression of retinol and lycopene (p<0.05 vs healthy). Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups (p<0.05 vs pre-supplementation) and attenuated the exercise-induced oxidative stress response (p<0.05 vs healthy). There were no selective treatment differences between groups in the primary outcome variable. Conclusions/ interpretation: These findings are the first to suggest that oral vitamin C supplementation provides an effective prophylaxis against exercise-induced free radical-mediated lipid peroxidation in human diabetic blood.

No clear evidence of an association between plasma concentrations of n-3 long-chain polyunsaturated fatty acids and depressed mood in a non-clinical population

Previous research suggests that low n-3 long-chain polyunsaturated fatty acid (n-3PUFA) status is associated with higher levels of depression in clinical populations. This analysis aimed to investigate the relationship between depressed mood and n-3PUFA status in a non-clinical population. The analysis was conducted on data collected as part of a large randomized controlled trial investigating the impact of n-3PUFA supplementation on depressed mood in a community-based population. On entry into the trial, data on depressed mood were collected using the Depression, Anxiety and Stress Scales (DASS) and the Beck Depression Inventory (BDI). Plasma concentrations of various n-3PUFAs and n-6 long-chain polyunsaturated fatty acids (n-6PUFAs) were obtained from fasting venous blood samples, and various demographics were also measured. Using regression, there was no evidence of an association between either measure of depressed mood and any of the measures of n-3PUFA status or of n-6PUFA: n-3PUFA ratios. Clear associations were also not found when demographic factors were included in the analyses. These findings suggest that n-3PUFAs may not have a role in the aetiology of minor depression. This is also consistent with the results of other studies that have not demonstrated an association between depressed mood and n-3PUFA status in non-clinical populations and epidemiological studies that have not demonstrated an association between depressed mood and n-3PUFA intake in these populations. (C) 2008 Elsevier Ltd. All rights reserved.

Vitamins C and E for prevention of pre-eclampsia in women with type 1 diabetes (DAPIT): a randomised placebo-controlled trial.

Background
Results of several trials of antioxidant use during pregnancy have not shown a reduction in pre-eclampsia, but the eff ect in women with diabetes is unknown. We aimed to assess whether supplementation with vitamins C and E reduced incidence of pre-eclampsia in women with type 1 diabetes.

Methods
We enrolled women from 25 UK antenatal metabolic clinics in a multicentre randomised placebo-controlled trial. Eligibility criteria were type 1 diabetes preceding pregnancy, presentation between 8 weeks’ and 22 weeks’ gestation, singleton pregnancy, and age 16 years or older. Women were randomly allocated in a 1:1 ratio to receive1000 mg vitamin C and 400 IU vitamin E (a-tocopherol) or matched placebo daily until delivery. The randomisation sequence was stratifi ed by centre with balanced blocks of eight patients. All trial personnel and participants were masked to treatment allocation. The primary endpoint was pre-eclampsia, which we defi ned as gestational hypertension with proteinuria. Analysis was by modifi ed intention to treat. This study is registered, ISRCTN27214045.

Findings
Between April, 2003, and June, 2008, 762 women were randomly allocated to treatment groups (379 vitamin supplementation, 383 placebo). The primary endpoint was assessed for 375 women allocated to receive vitamins, and 374 allocated to placebo. Rates of pre-eclampsia did not differ between vitamin (15%, n=57) and placebo (19%, 70)groups (risk ratio 0·81, 95% CI 0·59–1·12). No adverse maternal or neonatal outcomes were reported.

Interpretation
Supplementation with vitamins C and E did not reduce risk of pre-eclampsia in women with type 1 diabetes. However, the possibility that vitamin supplementation might be benefi cial in women with a low antioxidant status at baseline needs further testing.

The pitfall with PIT tags: Marking freshwater bivalves for translocation induces short-term behavioural costs

Tagging animals is frequently employed in ecological studies to monitor individual behaviour, for example postrelease survival and dispersal of captive-bred animals used in conservation programmes. While the majority of studies focus on the efficacy of tags in facilitating the relocation and identification of individuals, few assess the direct effects of tagging in biasing animal behaviour. We used an experimental approach with a control to differentiate the effects of handling and tagging captive-bred juvenile freshwater pearl mussels, Margaritifera margaritifera, prior to release into the wild. Marking individuals with passive integrated transponder (PIT) tags significantly decreased their burrowing rate and, therefore, increased the time taken to burrow into the substrate. This effect was contributed to, in part, by the detrimental impacts of handling, which also significantly affected activity, burrowing ability and the time taken for each individual to emerge and start probing the substrate. Disturbance during handling and tagging may lead to indirect mortality after release by increasing the risk of predation or dislodgement during flooding, thereby potentially compromising any conservation strategy contingent on population supplementation or reintroduction. This is the first study to demonstrate that handling and PIT tagging has a detrimental impact on invertebrate behaviour. Moreover, our results provide useful information that will inform freshwater bivalve conservation strategies.

Worldwide childhood type 1 diabetes incidence - what can we learn from epidemiology?

Type 1 diabetes is the most common form of diabetes in most part of the world, although reliable data are still unavailable in several countries. Wide variations exist between the incidence rates of different populations, incidence is lowest in China and Venezuela (0.1 per 100 000 per year) and highest in Finland and Sardinia (37 per 100 000 per year). In most populations girls and boys are equally affected. In general, the incidence increases with age, the incidence peak is at puberty. After the pubertal years, the incidence rate significantly drops in young women, but remains relatively high in young adult males up to the age 29-35 years. Prospective national and large international registries (DIAMOND and EURODIAB) demonstrated an increasing trend in incidence in most regions of the world over the last few decades and increases seem to be the highest in the youngest age group. Analytical epidemiological studies have identified environmental risk factors operating early in life which might have contributed to the increasing trend in incidence. These include enteroviral infections in pregnant women, older maternal age (39-42 years), preeclampsia, cesarean section delivery, increased birthweight, early introduction of cow's milk proteins and an increased rate of postnatal growth (weight and height). Optimal vitamin D supplementation during early life has been shown to be protective. Some of these environmental risk factors such as viruses may initiate autoimmunity toward the beta cell, other exposures may put on overload on the already affected beta cell and thus accelerate the disease process.

Applying species distribution modelling to identify areas of high conservation value for endangered species: A case study using Margaritifera margaritifera (L.)

The development and implementation of a population supplementation and restoration plan for any endangered species should involve an understanding of the species’ habitat requirements prior to the release of any captive bred individuals. The freshwater pearl mussel, Margaritifera margaritifera, has undergone dramatic declines over the last century and is now globally endangered. In Northern Ireland, the release of captive bred individuals is being used to support wild populations and repatriate the species in areas where it once existed. We employed a combination of maximum entropy modelling (MAXENT) and Generalized Linear Mixed Models (GLMM) to identify ecological parameters necessary to support wild populations using GIS-based landscape scale and ground-truthed habitat scale environmental parameters. The GIS-based landscape scale model suggested that mussel occurrence was associated with altitude and soil characteristics including the carbon, clay, sand, and silt content. Notably, mussels were associated with a relatively narrow band of variance indicating that M. margaritifera has a highly specific landscape niche. The ground-truthed habitat scale model suggested that mussel occurrence was associated with stable consolidated substrates, the extent of bankside trees, presence of indicative macrophyte species and fast flowing water. We propose a three phase conservation strategy for M. margaritifera identifying suitable areas within rivers that (i) have a high conservation value yet needing habitat restoration at a local level, (ii) sites for population supplementation of existing populations and (iii) sites for species reintroduction to rivers where the mussel historically occurred but is now locally extinct. A combined analytical approach including GIS-based landscape scale and ground-truthed habitat scale models provides a robust method by which suitable release sites can be identified for the population supplementation and restoration of an endangered species. Our results will be highly influential in the future management of M. margaritifera in Northern Ireland.

Folic acid use and major congenital malformations in offspring of women with epilepsy: a prospective study from the UK Epilepsy and Pregnancy Register

Objective: In the general population, folic acid supplementation during pregnancy has been demonstrated to reduce the frequency of neural tube defects (NTDs) and other major congenital malformations (MCMs). It is recommended that women with epilepsy contemplating pregnancy take supplemental folic acid because of the known antifolate effect of some antiepileptic drugs (AEDs). Here the aim was to determine the effectiveness of this practice.

Dose-dependent effects of folic acid on blood concentrations of homocysteine: a meta-analysis of the randomized trials

Background: Dietary supplementation with B vitamins that lower blood homocysteine concentrations is expected to reduce cardiovascular disease risk, but there has been uncertainty about the optimum regimen to use for this purpose.