Immuno-metabolic impact of the multiple sclerosis patients' sera on endothelial cells of the blood-brain barrier.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease which results from the invasion of the brain by activated immune cells across the endothelial cells (ECs) of the blood-brain barrier (BBB), due to loss of immune self-tolerance. Many reports define the metabolic profile of immune cells in MS, however little is known about the metabolism of the BBB ECs during the disease. We aim to determine whether circulating factors in MS induce metabolic alterations of the BBB ECs compared to a healthy state, which can be linked with disruption of BBB integrity and subsequent immune cell extravasation. METHODS AND RESULTS: In this report, we used an in vitro model to study the effect of sera from naïve-to-treatment, relapsing-remitting MS (RRMS) patients on the human brain microvascular endothelium, comparing effects to age/sex-matched healthy donor (HD) sera. Our data show that RRMS serum components affect brain endothelial cells by impairing intercellular tightness through the down-modulation of occludin and VE-cadherin, and facilitating immune cell extravasation through upregulation of intercellular adhesion molecules (ICAM-1) and P-glycoprotein (P-gp). At a metabolic level, the treatment of the endothelial cells with RRMS sera reduced their glycolytic activity (measured through the extracellular acidification rate-ECAR) and oxygen consumption rate (oxidative phosphorylation rate-OCR). Such changes were associated with the down-modulation of endothelial glucose transporter 1 (GLUT-1) expression and by altered mitochondrial membrane potential. Higher level of reactive oxygen species released from the endothelial cells treated with RRMS sera indicate a pro-inflammatory status of the cells together with the higher expression of ICAM-1, endothelial cell cytoskeleton perturbation (stress fibres) as well as disruption of the cytoskeleton signal transduction MSK1/2 and ß-catenin phosphorylation. CONCLUSIONS: Our data suggest that circulating factors present in RRMS patient serum induce physiological and biochemical alterations to the BBB, namely reducing expression of essential tightness regulators, as well as reduced engagement of glycolysis and alteration of mitochondrial potential. As these last changes have been linked with alterations in nutrient usage and metabolic function in immune cells; we propose that the BBB endothelium of MS patients may similarly undergo metabolic dysregulation, leading to enhanced permeability and increased disease susceptibility.

Contrasting Influences of Geographic Range and Distribution of Populations on Patterns of Genetic Diversity in Two Sympatric Pilbara Acacias.

The influence of geographic range on species persistence has long been of interest and there is a need for a better understanding of the genetic consequences for species with restricted distributions, particularly with the increasing rate of global species extinctions. However, the genetic effects of restricted range are often confounded by the impacts of population distribution. We compared chloroplast and nuclear genetic diversity and differentiation in two acacias, the restricted, patchily distributed Acacia atkinsiana and the widespread, semi-continuously distributed A. ancistrocarpa. Lower intra-population diversity and higher differentiation between populations were seen in A. atkinsiana compared to its widespread congener, A. ancistrocarpa. There was little evidence of geographical influences on population genetic structure in A. ancistrocarpa whereas A. atkinsiana exhibited nuclear genetic structure with isolation by distance, differentiation of near-coastal populations from those in the ranges, and differentiation of peripheral populations from those in the centre of the distribution. These results are consistent with expectations of the effect of geographic range and population distribution on genetic diversity, but indicate that distribution of populations rather than geographic range has influenced the observed genetic structure. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species.

Intervention improves assisted conception intracytoplasmic sperm injection outcomes for patients with high levels of sperm DNA fragmentation: a retrospective analysis.

Sperm DNA fragmentation (SDF) is used in assisted reproductive technology (ART) programs as an indicator for sperm quality, although there is still a lack of consensus as to its clinical utility. In this retrospective study, we examined intracytoplasmic sperm injection (ICSI) outcomes of 1924 infertile patients who underwent SDF analysis using the sperm chromatin integrity test. ART patients were classified as having low [DNA fragmentation index (DFI) <29%] or high SDF (DFI ≥29%) and by whether or not an intervention [physiological intracytoplasmic sperm injection (PICSI), intracytoplasmic morphologically selected sperm injection (IMSI), testicular sperm extraction (TESE)/testicular sperm aspiration (TESA), frequent ejaculation] was performed. High SDF patients who did not have an intervention had a lower fertilization rate and poorer clinical outcomes from blastocyst transfers as compared with low SDF patients; the fertilization rate was 66.0% vs. 70.2% (p = 0.042), single embryo transfer (SET) fetal heart pregnancy rate was 28.5% vs. 45.2% (p = 0.042), and SET live birth rate was 24.9% vs. 40.6% (p = 0.060), respectively. Furthermore, high SDF patients who had an intervention had significantly improved blastocyst transfer outcomes, similar to those of low SDF patients; the SET live birth rate for high SDF intervention patients was 43.8% as compared with 24.9% for high SDF no intervention patients (p = 0.037) and 40.6% for low SDF patients (p = 0.446). Analysis of the three main intervention subgroups for high SDF patients revealed that TESE/TESA patients had the highest SET live birth rate; in comparison with 24.2% for high SDF patients who did not have an intervention, PICSI patients had 38.3% (p = 0.151), IMSI patients had 28.7% (p = 0.680), and TESE/TESA patients had 49.8% (p = 0.020). Our data suggest that SDF results indicate ICSI outcomes and that patients who have high SDF benefit from an intervention.

Estrogen protects the blood-brain barrier from inflammation-induced disruption and increased lymphocyte trafficking.

Sex differences have been widely reported in neuroinflammatory disorders, focusing on the contributory role of estrogen. The microvascular endothelium of the brain is a critical component of the blood-brain barrier (BBB) and it is recognized as a major interface for communication between the periphery and the brain. As such, the cerebral capillary endothelium represents an important target for the peripheral estrogen neuroprotective functions, leading us to hypothesize that estrogen can limit BBB breakdown following the onset of peripheral inflammation. Comparison of male and female murine responses to peripheral LPS challenge revealed a short-term inflammation-induced deficit in BBB integrity in males that was not apparent in young females, but was notable in older, reproductively senescent females. Importantly, ovariectomy and hence estrogen loss recapitulated an aged phenotype in young females, which was reversible upon estradiol replacement. Using a well-established model of human cerebrovascular endothelial cells we investigated the effects of estradiol upon key barrier features, namely paracellular permeability, transendothelial electrical resistance, tight junction integrity and lymphocyte transmigration under basal and inflammatory conditions, modeled by treatment with TNFα and IFNγ. In all cases estradiol prevented inflammation-induced defects in barrier function, action mediated in large part through up-regulation of the central coordinator of tight junction integrity, annexin A1. The key role of this protein was then further confirmed in studies of human or murine annexin A1 genetic ablation models. Together, our data provide novel mechanisms for the protective effects of estrogen, and enhance our understanding of the beneficial role it plays in neurovascular/neuroimmune disease.

Sex-dependent diversity in ventral tegmental dopaminergic neurons and developmental programing: A molecular, cellular and behavioral analysis.

The knowledge that diverse populations of dopaminergic neurons within the ventral tegmental area (VTA) can be distinguished in terms of their molecular, electrophysiological and functional properties, as well as their differential projections to cortical and subcortical regions has significance for key brain functions, such as the regulation of motivation, working memory and sensorimotor control. Almost without exception, this understanding has evolved from landmark studies performed in the male sex. However, converging evidence from both clinical and pre-clinical studies illustrates that the structure and functioning of the VTA dopaminergic systems are intrinsically different in males and females. This may be driven by sex differences in the hormonal environment during adulthood ('activational' effects) and development (perinatal and/or pubertal 'organizational' effects), as well as genetic factors, especially the SRY gene on the Y chromosome in males, which is expressed in a sub-population of adult midbrain dopaminergic neurons. Stress and stress hormones, especially glucocorticoids, are important factors which interact with the VTA dopaminergic systems in order to achieve behavioral adaptation and enable the individual to cope with environmental change. Here, also, there is male/female diversity not only during adulthood, but also in early life when neurobiological programing by stress or glucocorticoid exposure differentially impacts dopaminergic developmental trajectories in male and female brains. This may have enduring consequences for individual resilience or susceptibility to pathophysiological change induced by stressors in later life, with potential translational significance for sex bias commonly found in disorders involving dysfunction of the mesocorticolimbic dopaminergic systems. These findings highlight the urgent need for a better understanding of the sexual dimorphism in the VTA if we are to improve strategies for the prevention and treatment of debilitating conditions which differentially affect men and women in their prevalence and nature, including schizophrenia, attention/deficit hyperactivity disorder, autism spectrum disorders, anxiety, depression and addiction.

Application of layer-by-layer coatings to tissue scaffolds - development of an angiogenic biomaterial.

Tissue engineered materials aimed at wound care typically underperform due to poor engrafting to the wound bed. The need for such materials will continue to intensify as a result of an ageing population and an increase in patients suffering from vascular problems. Here we describe the development of an angiogenic coating strategy employing a combination of plasma phase deposition of acrylic acid and layer-by-layer (LBL) chemistry using polyethyleneimine and poly(acrylic acid) for the immobilization of heparin and Vascular Endothelial Growth Factor (VEGF). The formation of the coating and its ability to immobilize heparin was examined by Quartz Crystal Microbalance with Dissipation. X-ray Photoelectron Spectroscopy (XPS) and Atomic Force Microscopy were used to confirm that these coatings retained a significant amount of heparin on the surface when applied to a flat substrate. The coating strategy was transferred to 2 different tissue scaffold architectures: a commercially available non-biodegradable polypropylene mesh, and a biodegradable electrospun poly(lactic-co-glycolic acid) (PLGA) scaffold. XPS confirmed that the coating was successfully applied to the scaffolds and that a similar amount of heparin was immobilized. In vitro testing showed that while HDMEC readily attached to the PLGA scaffold, they were inhibited from adhering and forming proliferative colonies where heparin alone was attached to the LBL coated PLGA scaffold. However, after dip coating with VEGF, the heparin coated scaffold supported both attachment and colony growth of HDMEC; no such colony formation occurred in the absence of VEGF.

The effect of metal microstructure on the initial attachment of Escherichia coli to 1010 carbon steel.

Metallurgical features have been shown to play an important role in the attachment of microorganisms to metal surfaces. In the present study, the influence of the microstructure of as-received (AR) and heat-treated (HT) 1010 carbon steel on the initial attachment of bacteria was investigated. Heat treatment was carried out with the aim of increasing the grain size of the carbon steel coupons. Mirror-polished carbon steel coupons were immersed in a minimal medium inoculated with Escherichia coli (ATCC 25922) to investigate the early (15, 30 and 60 min) and relatively longer-term (4 h) stages of bacterial attachment. The results showed preferential colonisation of bacteria on the grain boundaries of the steel coupons. The bacterial attachment to AR steel coupons was relatively uniform compared to the HT steel coupons where an increased number of localised aggregates of bacteria were found. Quantitative analysis showed that the ratio of the total number of isolated (i.e., single) bacteria to the number of bacteria in aggregates was significantly higher on the AR coupons than the HT coupons. Longer-term immersion studies showed production of extracellular polymeric substances by the bacteria and corrosion at the grain boundaries on both types of steel coupon tested.

Temperature measurement in the microscopic regime: a comparison between fluorescence lifetime- and intensity-based methods.

Thermally sensitive fluorescent indicators have been proposed to monitor temperature changes in microfluidic systems, mainly based on fluorescence intensity or lifetime. However, measuring temperature in a structured environment, such as biological tissue, presents additional challenges due to the chemical and structural complexity. Here, we investigate the potential for resolving temperature distributions within the volume of a single cell. Rhodamine B (RhB) dye was employed as a temperature indicator to compare fluorescence intensity- and lifetime-based techniques. The relationship between the fluorescence lifetime and temperature was found to be highly dependent on the biological environment. The intensity-based method allowed the temperature distribution to be mapped with partial success within the volume of a single cell. Under ideal circumstances, the temperature can be mapped pixel by pixel with a resolution better than ±0.3°C within the cell cytoplasm, but this accuracy was reduced to ±1.8°C by environmental variations. These results suggest that the fluorophore should be encapsulated and immobilized in the biological tissue in order to reduce the influence of environmental factors on temperature measurements at the cellular level.

Stable low-fouling plasma polymer coatings on polydimethylsiloxane.

Polydimethylsiloxane (DMS) is a popular material for microfluidics, but it is hydrophobic and is prone to non-specific protein adsorption. In this study, we explore methods for producing stable, protein resistant, tetraglyme plasma polymer coatings on PDMS by combining extended baking processes with multiple plasma polymer coating steps. We demonstrate that by using this approach, it is possible to produce a plasma polymer coatings that resist protein adsorption (<10 ng/cm(2)) and are stable to storage over at least 100 days. This methodology can translate to any plasma polymer system, enabling the introduction of a wide range of surface functionalities on PDMS surfaces.

Blastocyst trophectoderm biopsy and preimplantation genetic diagnosis for familial monogenic disorders and chromosomal translocations.

OBJECTIVE: Modern in vitro fertilization practices involve transfer of embryos as blastocysts, when anabolic metabolism is well established and pregnancy rates can be maintained while transferring embryos singly to avoid multiple pregnancies. Embryo biopsy for preimplantation genetic diagnosis (PGD), however, is generally performed on day 3, when the embryo comprises just 6 to 8 cells, one or two of which are removed for testing. Implantation rates and clinical pregnancy rates have remained relatively low and a harmful effect from losing one or more cells from such early embryos has not been excluded. METHODS: We performed a sequential study involving 399 egg retrievals and 1879 embryo biopsies for patients undergoing PGD to avoid a serious monogenic disease or an unbalanced chromosomal translocation. We compared implantation and viable pregnancy rates after biopsies taken on day 3 (cleavage-stage biopsy) with biopsies delayed until day 5 or 6, when the embryo is a blastocyst and 5 or more cells can be sampled from the trophectoderm while the inner cell mass, from which the fetus develops, remains intact. All embryos were transferred as blastocysts. RESULTS: Despite fewer blastocysts than cleavage embryos biopsied and tested (3.6 compared to 6.6), implantation rates per embryo transferred were 43.4% if biopsied at the blastocyst stage and 25.6% if biopsied at the cleavage stage (P < 0.01), with ongoing or live-birth pregnancy rates per egg retrieval of 34.2% (average transfer number 1.1) for blastocyst biopsies and 25.5% (transfer number 1.6) for cleavage stage biopsies (P < 0.05, 1-tailed). The multiple pregnancy rate for monogenic disease exclusion fell from 16.7% to 2% (P = 0.04, 1-tailed). CONCLUSIONS: For exclusion of genetic disease, day 5-6 blastocyst-stage biopsies are more likely to be followed by implantation and singleton births than is the case after PGD performed on day 3.

Perinatal glucocorticoid treatment disrupts the hypothalamo-lactotroph axis in adult female, but not male, rats.

This study aimed to test the hypothesis that the tuberoinfundibular dopaminergic neurons of the arcuate nucleus and/or the lactotroph cells of the anterior pituitary gland are key targets for the programming effects of perinatal glucocorticoids (GCs). Dexamethasone was administered noninvasively to fetal or neonatal rats via the mothers' drinking water (1 mug/ml) on embryonic d 16-19 or neonatal d 1-7, and control animals received normal drinking water. At 68 d of age, the numbers of tyrosine hydroxylase-positive (TH+) cells in the arcuate nucleus and morphometric parameters of pituitary lactotrophs were analyzed. In control animals, striking sex differences in TH+ cell numbers, lactotroph cell size, and pituitary prolactin content were observed. Both pre- and neonatal GC treatment regimens were without effect in adult male rats, but in females, the overriding effect was to abolish the sex differences by reducing arcuate TH+ cell numbers (pre- and neonatal treatments) and reducing lactotroph cell size and pituitary prolactin content (prenatal treatment only) without changing lactotroph cell numbers. Changes in circulating prolactin levels represented a net effect of hypothalamic and pituitary alterations that exhibited independent critical windows of susceptibility to perinatal GC treatments. The dopaminergic neurons of the hypothalamic periventricular nucleus and the pituitary somatotroph populations were not significantly affected by either treatment regimen in either sex. These data show that the adult female hypothalamo-lactotroph axis is profoundly affected by perinatal exposure to GCs, which disrupts the tonic inhibitory tuberoinfundibular dopaminergic pathway and changes lactotroph morphology and prolactin levels in the pituitary and circulation. These findings provide new evidence for a long-term disruption in prolactin-dependent homeostasis in females, but not males, after inappropriate GC exposure in perinatal life.

Altered mesencephalic dopaminergic populations in adulthood as a consequence of brief perinatal glucocorticoid exposure.

Early exposure to stressors is strongly associated with enduring effects on central nervous system function, but the mechanisms and neural substrates involved in this biological 'programming' are unclear. This study tested the hypothesis that inappropriate exposure to glucocorticoid stress hormones (GCs) during critical periods of development permanently alters the mesencephalic dopaminergic populations in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Using a rat model, the synthetic GC dexamethasone was added to the maternal drinking water during gestational days 16-19 or over the first week of postnatal life. In adulthood, the effects upon tyrosine hydroxylase immunopositive (TH+) cell numbers in the midbrain, and monoamine levels in the forebrain, of the adult offspring were assessed and compared with control offspring whose dams received normal drinking water. In the VTA, both prenatal and postnatal dexamethasone treatment increased TH+ cell numbers by approximately 50% in males and females. Although prenatal dexamethasone treatment also increased TH+ cell numbers in the SNc by 40-50% in males and females, postnatal treatment affected females only by increasing TH+ cell numbers by approximately 30%. In comparison, similar changes were not detected in the monoamine levels of the dorsolateral striatum, nucleus accumbens or infralimbic cortex of either males or females, which is a feature likely to reflect adaptive changes in these pathways. These studies demonstrate that the survival or phenotypic expression of VTA and SNc dopaminergic neurones is profoundly influenced by brief perinatal exposure to GCs at times when endogenous levels are normally low. These findings are the first to demonstrate permanent changes in the cytoarchitecture within midbrain dopamine nuclei after perinatal exposure to stress hormones and implicate altered functionality. Thus, they have significance for the increasing use of GCs in perinatal medicine and indicate potential mechanisms whereby perinatal distress may predispose to the development of a range of psychiatric conditions in later life.

Surface-MALDI mass spectrometry in biomaterials research.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.

Dose- and sex-dependent effects of the neurotoxin 6-hydroxydopamine on the nigrostriatal dopaminergic pathway of adult rats: differential actions of estrogen in males and females.

Epidemiological and clinical studies provide growing evidence for marked sex differences in the incidence of certain neurological disorders that are largely attributed to the neuroprotective effects of estrogen. Thus there is a keen interest in the clinical potential of estrogen-related compounds to act as novel therapeutic agents in conditions of neuronal injury and neurodegeneration such as Parkinson's disease. Studies employing animal models of neurodegeneration in ovariectomised female rats treated with estrogen support this hypothesis, yet experimental evidence for sex differences in the CNS response to direct neurotoxic insult is limited and, as yet, few studies have addressed the role played by endogenously produced hormones in neuroprotection. Therefore, in this study we aimed to determine (1) whether the prevailing levels of sex steroid hormones in the intact rat provide a degree of protection against neuronal assault in females compared with males and (2) whether sex differences depend solely on male/female differences in circulating estrogen levels or whether androgens could also play a role. Using the selective, centrally administered neurotoxin 6-hydroxydopamine, which induces a lesion in the nigrostriatal dopaminergic pathway similar to that seen in Parkinson's disease, we have demonstrated a sexually dimorphic (male-dominant), dose-dependent susceptibility in rats. Furthermore, following gonadectomy, dopamine depletion resulting from a submaximal dose of 6-hydroxydopamine (1 microg) was reduced in male rats, whereas in females, ovariectomy enhanced dopamine depletion. Administration of the nonaromatizable androgen dihydrotestosterone to gonadectomized animals had no significant effect on 6-hydroxydopamine toxicity in either males or females, whereas treatment of gonadectomized males and females with physiological levels of estrogen restored the extent of striatal dopamine loss to that seen in intact rats, viz, estrogen therapy reduced lesion size in females but increased it in males. Taken together, our findings strongly suggest that there are sex differences in the mechanisms whereby nigrostriatal dopaminergic neurones respond to injury. They also reveal that the reported clinically beneficial effects of estrogen in females may not be universally adopted for males. While the reasons for this gender-determined difference in response to the activational action of estrogen are unknown, we hypothesize that they may well be related to the early organizational events mediated by sex steroid hormones, which ultimately result in the sexual differentiation of the brain.

XPS and surface-MALDI-MS characterisation of worn HEMA-based contact lenses.

XPS and MALDI-MS were used to analyse initial adsorption events in the fouling of HEMA-based contact lenses. All of the lenses tested accumulated tear film deposits within 10 min of wear. XPS indicated the presence of mainly proteinaceous deposits, with indications of some contributions by mucins or lipids on some lenses and the nature of the deposit being influenced by the lens chemistry. MALDI-MS detected the presence of surface-adsorbed species with molecular weights < 15 kDa. While lysozyme could be identified by comparison of MALDI-MS signals with known protein mass and assignments are suggested for some other signals, several other species, with MWs less than that of lysozyme, could not be identified as no ocular proteins with corresponding MWs had been reported in previous biochemical tear film analyses. These species, and others, were also detected in MALDI-MS analysis of reflex tear film, suggesting that the adsorbed unidentified species were not simply products of surface-induced dissociation of adsorbing higher-MW proteins. This short-term wear study detected rapid interface conversion and demonstrated the utility and surface sensitivity of XPS and MALDI-MS in characterising contact lens deposits at the initial stages when sub-monolayer adsorbed amounts are present on lenses.