Relation between neuronal morphology and electrophysiology in the Kainate lesion model of Alzheimer's Disease

This paper describes a computational and statistical study of the influence of morphological changes on the electrophysiological response of neurons from an animal model of Alzheimer's Disease (AD). We combined experimental morphological data from rat hippocampal CA1 pyramidal cells with a well-established model of active membrane properties. Dendritic morphology and the somatic response to simulated current clamp conditions were then compared for cells from the control and the AD group. The computational approach allowed us to single out the influences of neuromorphology on neuronal response by eliminating the effects of active channel variability. The results did not reveal a simple relationship between morphological changes associated with AD and changes in neural response. However, they did suggest the existence of more complex than anticipated relationships between dendritic morphology and single-cell electrophysiology.

Diallyl disulphide, a beneficial component of garlic oil, causes a redistribution of cell-cycle growth phases, induces apoptosis and enhances butyrate-induced apoptosis in colorectal adenocarcinoma cells (HT-29)

Colon cancer is a leading and expanding cause of death worldwide. A major contributory factor to this disease is diet composition; some components are beneficial (e.g. dietary fibre) whilst others are detrimental (e.g. alcohol). Garlic oil is a prominent dietary constituent that prevents the development of colorectal cancer. This effect is believed to be mainly due to diallyl disulphide (DADS), which selectively induces redox stress in cancerous (rather than normal) cells which leads to apoptotic cell death. However, the detailed mechanism by which DADS causes apoptosis remains unclear. We show that DADS-treatment of colonic adenocarcinoma cells (HT-29) initiates a cascade of molecular events characteristic of apoptosis. These include a decrease in cellular proliferation, translocation of phosphatidylserine to the plasma-membrane outer-layer, activation of caspase-3, genomic-DNA fragmentation and G2/M phase cell-cycle arrest. Short-chain fatty acids (SCFAs), particularly butyrate (abundantly produced in the gut by bacterial fermentation of dietary polysaccharides), enhance colonic cell integrity but, in contrast, inhibit colonic-cancer cell growth. Combining DADS with butyrate augmented the effect of butyrate on HT-29 cells. These results suggest that the anti-cancerous properties of DADS afford greater benefit when supplied with other favourable dietary factors (SCFA/polysaccharides) that likewise reduce colonic tumour development.

Riluzole neuroprotection in a parkinson’s disease model involves suppression of reactive astrocytosis but not GLT-1 regulation

Background Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson's disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson’s disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

TRIM32 regulates skeletal muscle stem cell differentiation and is necessary for normal adult muscle regeneration

Limb girdle muscular dystrophy type 2H (LGMD2H) is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Currently its pathogenesis is entirely unclear. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian neural progenitor cells through controlling their differentiation, we asked whether TRIM32 could also be essential for the regulation of myogenic stem cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, we show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly we show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. Our studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Ethnic differences in beta-cell function, dietary intake and expression of the metabolic syndrome among UK adults of south Asian, black African-Caribbean and white-European origin at high risk of metabolic syndrome

A cross-sectional analysis of ethnic differences in dietary intake, insulin sensitivity and beta-cell function, using the intravenous glucose tolerance test (IVGTT), was conducted on 497 healthy adult participants of the ‘Reading, Imperial, Surrey, Cambridge, and Kings’ (RISCK) study. Insulin sensitivity (Si) was significantly lower in African-Caribbean (AC) and South Asian (SA) participants [IVGTT-Si; AC: 2.13 vs SA: 2.25 vs white-European (WE): 2.84 (×10−4 mL µU min)2, p < 0.001]. AC participants had a higher prevalence of anti-hypertensive therapy (AC: 19.7% vs SA: 7.5%), the most cardioprotective lipid profile [total:high-density lipoprotein (HDL); AC: 3.52 vs SA: 4.08 vs WE: 3.83, p = 0.03] and more pronounced hyperinsulinaemia [IVGTT–acute insulin response (AIR)] [AC: 575 vs SA: 428 vs WE: 344 mL/µU/min)2, p = 0.002], specifically in female participants. Intake of saturated fat and carbohydrate was lower and higher in AC (10.9% and 50.4%) and SA (11.1% and 52.3%), respectively, compared to WE (13.6% and 43.8%, p < 0.001). Insulin resistance in ACs is characterised by ‘normal’ lipid profiles but high rates of hypertension and pronounced hyperinsulinaemia.

The long-term safety and efficacy of bilateral transplantation of human fetal striatal tissue in patients with mild to moderate Huntington's disease.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease involving progressive motor, cognitive and behavioural decline, leading to death approximately 20 years after motor onset. The disease is characterised pathologically by an early and progressive striatal neuronal cell loss and atrophy, which has provided the rationale for first clinical trials of neural repair using fetal striatal cell transplantation. Between 2000 and 2003, the 'NEST-UK' consortium carried out bilateral striatal transplants of human fetal striatal tissue in five HD patients. This paper describes the long-term follow up over a 3-10-year postoperative period of the patients, grafted and non-grafted, recruited to this cohort using the 'Core assessment program for intracerebral transplantations-HD' assessment protocol. No significant differences were found over time between the patients, grafted and non-grafted, on any subscore of the Unified Huntington's Disease Rating Scale, nor on the Mini Mental State Examination. There was a trend towards a slowing of progression on some timed motor tasks in four of the five patients with transplants, but overall, the trial showed no significant benefit of striatal allografts in comparison with a reference cohort of patients without grafts. Importantly, no significant adverse or placebo effects were seen. Notably, the raclopride positron emission tomography (PET) signal in individuals with transplants, indicated that there was no obvious surviving striatal graft tissue. This study concludes that fetal striatal allografting in HD is safe. While no sustained functional benefit was seen, we conclude that this may relate to the small amount of tissue that was grafted in this safety study compared with other reports of more successful transplants in patients with HD.

Human microbiota in health and disease

Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease. Studies in monozygotic twins, showing that these retain highly similar microbiota decades after birth and initial colonisation, are strongly indicative that diversity of the microbiome is host-specific and affected by the genotype. Microbial diversity in the human body is reflected in both richness and evenness. Diversity increases steeply from birth reaching its highest point in early adulthood, before declining in older age. However, in healthy subjects there appears to be a core of microbial phylotypes which remains relatively stable over time. Studies of individuals from diverse geopraphies suggest that clusters of intestinal bacterial groups tend to occur together, constituting ‘enterotypes’. So variation in intestinal microbiota is stratified rather than continuous and there may be a limited number of host/microbial states which respond differently to environmental influences. Exploration of enterotypes and functional groups may provide biomarkers for disease and insights into the potential for new treatments based on manipulation of the microbiome. In health, the microbiota interact with host defences and exist in harmonious homeostasis which can then be disturbed by invading organisms or when ‘carpet bombing’ by antibiotics occurs. In a portion of individuals with infections, the disease will resolve itself without the need for antibiotics and microbial homeostasis with the host’s defences is restored. The administration of probiotics (live microorganisms which when administered in adequate amounts confer a health benefit on the host) represents an artificial way to enhance or stimulate these natural processes. The study of innate mechanisms of antimicrobial defence on the skin, including the production of numerous antimicrobial peptides (AMPs), has shown an important role for skin commensal organisms. These organisms may produce AMPs, and also amplify the innate immune responses to pathogens by activating signalling pathways and processing host produced AMPs. Research continues into how to enhance and manipulate the role of commensal organisms on the skin. The challenges of skin infection (including diseases caused by multiply resistant organisms) and infestations remain considerable. The potential to re-colonise the skin to replace or reduce pathogens, and exploring the relationship between microbiota elsewhere and skin diseases are among a growing list of research targets. Lactobacillus species are among the best known ‘beneficial’ bacterial members of the human microbiota. Of the approximately 120 species known, about 15 are known to occur in the human vagina. These organisms have multiple properties, including the production of lactic acid, hydrogen peroxide and bacteriocins, which render the vagina inhospitable to potential pathogens. Depletion of the of the normal Lactobacillus population and overgrowth of vaginal anaerobes, accompanied by the loss of normal vaginal acidity can lead to bacterial vaginosis – the commonest cause of abnormal vaginal discharge in women. Some vaginal anaerobes are associated with the formation of vaginal biofilms which serve to act as a reservoir of organisms which persists after standard antibiotic therapy of bacterial vaginosis and may help to account for the characteristically high relapse rate in the condition. Administration of Lactobacillus species both vaginally and orally have shown beneficial effects in the treatment of bacterial vaginosis and such treatments have an excellent overall safety record. Candida albicans is a frequent coloniser of human skin and mucosal membranes, and is a normal part of the microbiota in the mouth, gut and vagina. Nevertheless Candida albicans is the most common fungal pathogen worldwide and is a leading cause of serious and often fatal nosocomial infections. What turns this organism from a commensal to a pathogen is a combination of increasing virulence in the organism and predisposing host factors that compromise immunity. There has been considerable research into the use of probiotic Lactobacillus spp. in vaginal candidiasis. Studies in reconstituted human epithelium and monolayer cell cultures have shown that L. rhamnosus GG can protect mucosa from damage caused by Candida albicans, and enhance the immune responses of mucosal surfaces. Such findings offer the promise that the use of such probiotic bacteria could provide new options for antifungal therapy. Studies of changes of the human intestinal microbiota in health and disease are complicated by its size and diversity. The Alimentary Pharmabiotic Centre in Cork (Republic of Ireland) has the mission to ‘mine microbes for mankind’ and its work illustrates the potential benefits of understanding the gut microbiota. Work undertaken at the centre includes: mapping changes in the microbiota with age; studies of the interaction between the microbiota and the gut; potential interactions between the gut microbiota and the central nervous system; the potential for probiotics to act as anti-infectives including through the production of bacteriocins; and the characterisation of interactions between gut microbiota and bile acids which have important roles as signalling molecules and in immunity. The important disease entity where the role of the gut microbiota appears to be central is the Irritable Bowel Syndrome (IBS). IBS patients show evidence of immune activation, impaired gut barrier function and abnormal gut microbiota. Studies with probiotics have shown that these organisms can exert anti-inflammatory effects in inflammatory bowel disease and may strengthen the gut barrier in IBS of the diarrhoea-predominant type. Formal randomised trials of probiotics in IBS show mixed results with limited benefit for some but not all. Studies confirm that administered probiotics can survive and temporarily colonise the gut. They can also stimulate the numbers of other lactic acid bacilli in the gut, and reduce the numbers of pathogens. However consuming live organisms is not the only way to influence gut microbiota. Dietary prebiotics are selectively fermented ingredients that can change the composition and/or activity of the gastrointestinal microbiota in beneficial ways. Dietary components that reach the colon, and are available to influence the microbiota include poorly digestible carbohydrates, such as non-starch polysaccharides, resistant starch, non-digestible oligosaccharides (NDOs) and polyphenols. Mixtures of probiotic and prebiotic ingredients that can selectively stimulate growth or activity of health promoting bacteria have been termed ‘synbiotics’. All of these approaches can influence gut microbial ecology, mainly to increase bifidobacteria and lactobacilli, but metagenomic approaches may reveal wider effects. Characterising how these changes produce physiological benefits may enable broader use of these tactics in health and disease in the future. The current status of probiotic products commercially available worldwide is less than ideal. Prevalent problems include misidentification of ingredient organisms and poor viability of probiotic microorganisms leading to inadequate shelf life. On occasions these problems mean that some commercially available products cannot be considered to meet the definition of a probiotic product. Given the potential benefits of manipulating the human microbiota for beneficial effects, there is a clear need for improved regulation of probiotics. The potential importance of the human microbiota cannot be overstated. ‘We feed our microbes, they talk to us and we benefit. We just have to understand and then exploit this.’ (Willem de Vos).

Investigating flavonoids as molecular templates for the design of small-molecule inhibitors of cell signaling

Epidemiological and clinical trials reveal compelling evidence for the ability of dietary flavonoids to lower cardiovascular disease risk. The mechanisms of action of these polyphenolic compounds are diverse, and of particular interest is their ability to function as protein and lipid kinase inhibitors. We have previously described structure-activity studies that reinforce the possibility for using flavonoid structures as templates for drug design. In the present study, we aim to begin constructing rational screening strategies for exploiting these compounds as templates for the design of clinically relevant, antiplatelet agents. We used the platelet as a model system to dissect the structural influence of flavonoids, stilbenes, anthocyanidins, and phenolic acids on inhibition of cell signaling and function. Functional groups identified as relevant for potent inhibition of platelet function included at least 2 benzene rings, a hydroxylated B ring, a planar C ring, a C ring ketone group, and a C-2 positioned B ring. Hydroxylation of the B ring with either a catechol group or a single C-4' hydroxyl may be required for efficient inhibition of collagen-stimulated tyrosine phosphorylated proteins of 125 to 130 kDa, but may not be necessary for that of phosphotyrosine proteins at approximately 29 kDa. The removal of the C ring C-3 hydroxyl together with a hydroxylated B ring (apigenin) may confer selectivity for 37 to 38 kDa phosphotyrosine proteins. We conclude that this study may form the basis for construction of maps of flavonoid inhibitory activity on kinase targets that may allow a multitargeted therapeutic approach with analogue counterparts and parent compounds.

Targeting stem cell niches and trafficking for cardiovascular therapy.

Regenerative cardiovascular medicine is the frontline of 21st-century health care. Cell therapy trials using bone marrow progenitor cells documented that the approach is feasible, safe and potentially beneficial in patients with ischemic disease. However, cardiovascular prevention and rehabilitation strategies should aim to conserve the pristine healing capacity of a healthy organism as well as reactivate it under disease conditions. This requires an increased understanding of stem cell microenvironment and trafficking mechanisms. Engagement and disengagement of stem cells of the osteoblastic niche is a dynamic process, finely tuned to allow low amounts of cells move out of the bone marrow and into the circulation on a regular basis. The balance is altered under stress situations, like tissue injury or ischemia, leading to remarkably increased cell egression. Individual populations of circulating progenitor cells could give rise to mature tissue cells (e.g. endothelial cells or cardiomyocytes), while the majority may differentiate to leukocytes, affecting the environment of homing sites in a paracrine way, e.g. promoting endothelial survival, proliferation and function, as well as attenuating or enhancing inflammation. This review focuses on the dynamics of the stem cell niche in healthy and disease conditions and on therapeutic means to direct stem cell/progenitor cell mobilization and recruitment into improved tissue repair.

Comparative effect of dairy fatty acids on cell adhesion molecules, nitric oxide and relative gene expression in healthy and diabetic human aortic endothelial cells

Dairy intake, despite its high saturated fatty acid (SFA) content, is associated with a lower risk of cardiovascular disease and diabetes. This in vitro study determined the effect of individual fatty acids (FA) found in dairy, and FA mixtures representative of a high SFA and a low SFA dairy lipid on markers of endothelial function in healthy and type II diabetic aortic endothelial cells.

Flavonoid-rich fruits and vegetables improve microvascular reactivity and inflammatory status in men at risk of cardiovascular disease – FLAVURS: a randomized controlled trial

BACKGROUND: Observed associations between increased fruit and vegetable (F&V) consumption, particularly those F&Vs that are rich in flavonoids, and vascular health improvements require confirmation in adequately powered randomized controlled trials. OBJECTIVE: This study was designed to measure the dose-response relation between high-flavonoid (HF), low-flavonoid (LF), and habitual F&V intakes and vascular function and other cardiovascular disease (CVD) risk indicators. DESIGN: A single-blind, dose-dependent, parallel randomized controlled dietary intervention study was conducted. Male and female low-F&V consumers who had a ≥1.5-fold increased risk of CVD (n = 174) were randomly assigned to receive an HF F&V, an LF F&V, or a habitual diet, with HF and LF F&V amounts sequentially increasing by 2, 4, and 6 (+2, +4, and +6) portions/d every 6 wk over habitual intakes. Microvascular reactivity (laser Doppler imaging with iontophoresis), arterial stiffness [pulse wave velocity, pulse wave analysis (PWA)], 24-h ambulatory blood pressure, and biomarkers of nitric oxide (NO), vascular function, and inflammation were determined at baseline and at 6, 12, and 18 wk. RESULTS: In men, the HF F&V diet increased endothelium-dependent microvascular reactivity (P = 0.017) with +2 portions/d (at 6 wk) and reduced C-reactive protein (P = 0.001), E-selectin (P = 0.0005), and vascular cell adhesion molecule (P = 0.0468) with +4 portions/d (at 12 wk). HF F&Vs increased plasma NO (P = 0.0243) with +4 portions/d (at 12 wk) in the group as a whole. An increase in F&Vs, regardless of flavonoid content in the groups as a whole, mitigated increases in vascular stiffness measured by PWA (P = 0.0065) and reductions in NO (P = 0.0299) in the control group. CONCLUSION: These data support recommendations to increase F&V intake to ≥6 portions daily, with additional benefit from F&Vs that are rich in flavonoids, particularly in men with an increased risk of CVD. This trial was registered at www.controlled-trials.com as ISRCTN47748735.