Dichloroacetate (DCA) reduces brain lactate but increases brain glutamine in experimental cerebral malaria: a 1H-NMR study

Abstract

Recent findings that levels of brain lactate and alanine were elevated in murine cerebral malaria led us to investigate the effect of dichloroacetate (DCA; 60 mg/kg), an activator of pyruvate dehydrogenase, on the levels of brain metabolites, and on the survival of mice infected with Plasmodium berghei ANKA which normally causes lethal cerebral malaria. DCA significantly reduced brain lactate and alanine levels when administered to infected mice, had no effect on the TCA cycle-related metabolites glutamate, GABA and aspartate and was associated with increased brain glutamine levels: 40% of mice thus treated survived the normally lethal infection.     

Inhaled nitric oxide reduces endothelial activation and parasite accumulation in the brain, and enhances survival in experimental cerebral malaria

The host immune response contributes to the onset and progression of severe malaria syndromes, such as cerebral malaria. Adjunctive immunomodulatory strategies for severe malaria may improve clinical outcome beyond that achievable with artemisinin-based therapy alone. Here, we report that prophylaxis with inhaled nitric oxide significantly reduced systemic inflammation (lower TNF, IFNγ and MCP-1 in peripheral blood) and endothelial activation (decreased sICAM-1 and vWF, and increased angiopoeitin-1 levels in peripheral blood) in an experimental cerebral malaria model. Mice that received inhaled nitric oxide starting prior to infection had reduced parasitized erythrocyte accumulation in the brain, decreased brain expression of ICAM-1, and preserved vascular integrity compared to control mice.Inhaled nitric oxide administered in combination with artesunate, starting as late as 5.5 days post-infection, improved survival over treatment with artesunate alone (70% survival in the artesunate only vs. 100% survival in the artesunate plus iNO group, p = 0.03). These data support the clinical investigation of inhaled nitric oxide as a novel adjunctive therapy in patients with severe malaria.     

Localized 1H-NMR spectroscopy in patients with fibromyalgia: a controlled study of changes in cerebral glutamate/glutamine, inositol, choline, and N-acetylaspartate

Introduction  

The purpose of this study was to investigate whether single-voxel (SV) proton magnetic resonance spectroscopy (MRS), diffusion-weighted imaging (DWI), and diffusion tensor imaging (DTI) detected differences between fibromyalgia (FM) patients and healthy controls. We also searched for correlations between neuroimaging abnormalities and neuropsychological variables.     

Farnesol-DMPC phase behaviour: a (2)H-NMR study

Involved in a number of diverse metabolic and functional contexts, farnesol is a central component of the mevalonate pathway, post-translationally attaches to proteins, and affects a number of other membrane-associated events. Despite farnesol's biological implications, a detailed analysis of how farnesol affects the physical properties and phase behaviour of lipid membranes is lacking. As (2)H-NMR spectra are sensitive to molecular motions and acyl chain orientation, they can be used to measure the degree of molecular order present in the system. Also, since the (2)H-NMR spectra of fluid and gel phase lipids are very different, they are sensitive probes of membrane phase equilibrium and can be used to determine fluid-gel phase boundaries. In this study, dimyristoyl phosphatidylcholine-d(54) (DMPC-d(54)) bilayers containing varying concentrations of trans-trans farnesol (2.5-20.0 mol%) are investigated over a range of temperatures (8-30 degrees C). Analysis of these spectra has led to the construction of a farnesol-DMPC-d(54) temperature-composition plot. We show that increasing concentrations of farnesol induce a decrease in the fluid-gel phase transition temperature and promote fluid-gel coexistence. Interestingly, farnesol does not seem to affect the quadrupolar splittings (Delta v(Q)) in the fluid phase, i.e., the organization of farnesol within the bilayer and its interaction with phospholipids does not appreciably influence acyl chain order in the fluid phase.     

Oil pollution increases plasma antioxidants but reduces coloration in a seabird

It has been suggested that condition-dependent signals may be a useful measure of environmental quality. In this study, we tested the hypothesis that oil pollution enhances oxidative stress and impairs expression of a carotenoid-based signal in a wild population of the yellow-legged gull (Larus michahellis). During the courtship period, a group of gulls were fed a supplementary diet containing heavy fuel oil from the Prestige oil spill and were compared with control gulls fed a similar supplementary diet without fuel oil. Blood levels of polycyclic aromatic hydrocarbons, the most toxic components of crude oils, were higher (30%) in the Prestige oil-fed gulls than in the control gulls. Plasma concentrations of vitamin E and carotenoids were also significantly higher in the Prestige oil-fed gulls (31 and 27%, respectively). Although, the plasma levels of lipid peroxidation markers were higher (13%) in gulls fed with Prestige oil than in the control gulls, these differences were not significant, possibly because of the small number of gulls analyzed. The red bill spot was significantly smaller (16%) in the oil-fed gulls than in the control individuals. This study provides the first experimental evidence that a carotenoid-based signal in a free-living seabird is affected by exposure to oil pollution and is hence indicative of environmental quality. Since the yellow-legged gull belongs to a complex of species widely distributed throughout the northern hemisphere, the method described may constitute a useful tool for evaluating sub-lethal effects of oil spills in seabirds.     

Dichloroacetate (DCA) enhances tumor cell death in combination with oncolytic adenovirus armed with MDA-7/IL-24

Dichloroacetate (DCA) is a metabolic modulator for the treatment of lactic acidosis and inherited mitochondrial diseases. A recent study showed that DCA treatment could induce apoptosis in many kinds of tumor cell lines via mitochondrial apoptotic pathway while sparing normal cells. ONYX-015 (dl 1520) is one of the oncolytic adenoviruses developed by the deletion of E1B-55kD gene of type 5 adenoviral DNA, and it replicates efficiently and selectively in tumor cells. ZD55-IL-24, an E1B-55kD deleted oncolytic adenovirus carrying interleukin-24 (IL-24, also called melanoma differentiation associated gene-7), had showed potent antitumor efficacy in a variety of tumor cells and exerted no apparent toxicity on normal cells. Given both the good therapeutic effect and low toxicity of these agents, here we investigated whether DCA in combination with ZD55-IL-24 or ONYX-015 could have more efficient antitumor activity in vitro experiments. Therefore, we tested the cytotoxicity of combination therapy in normal hepatic cells L-02 and QSG-7701 using the MTT assay. Our results showed that DCA combined with ONYX-015 or ZD55-IL-24 exhibited more potent antitumor activity than DCA or virus alone, and the combination treatment did not have superimposed toxicities in normal cells. Thus, a novel combination therapy associating oncolytic adenoviruses with relatively low toxic drug without severe side effects was proposed.     

S1P is associated with protection in human and experimental cerebral malaria

Cerebral malaria (CM) is associated with excessive inflammatory responses and endothelial activation. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid implicated in regulating vascular integrity, inflammation and T-cell migration. We hypothesized that altered S1P signaling during malaria contributes to endothelial activation and inflammation, and show that plasma S1P levels were decreased in Ugandan children with CM compared with children with uncomplicated malaria. Using the Plasmodium berghei ANKA (PbA) model of experimental CM (ECM), we demonstrate that humanized S1P lyase (hS1PL)(-/-) mice with reduced S1P lyase activity (resulting in increased bio-available S1P) had improved survival compared with wild-type littermates. Prophylactic and therapeutic treatment of infected mice with compounds that modulate the S1P pathway and are in human trials for other conditions (FTY720 or LX2931) significantly improved survival in ECM. FTY720 treatment improved vascular integrity as indicated by reduced levels of soluble intercellular adhesion molecule (sICAM), increased angiopoietin 1 (Ang1) (regulator of endothelial quiescence) levels, and decreased Evans blue dye leakage into brain parenchyma. Furthermore, treatment with FTY720 decreased IFNγ levels in plasma as well as CD4(+) and CD8(+) T-cell infiltration into the brain. Finally, when administered during infection in combination with artesunate, FTY720 treatment resulted in increased survival to ECM. These findings implicate dysregulation of the S1P pathway in the pathogenesis of human and murine CM and suggest a novel therapeutic strategy to improve clinical outcome in severe malaria.     

Coffee component 3-caffeoylquinic acid increases antioxidant capacity but not polyphenol content in experimental cerebral infarction

Although coffee has antioxidant capacity, it is not known which of its bioactive compounds is responsible for it, nor has it been analyzed in experimental cerebral infarction. We studied the effect one of its compounds, 3-caffeoylquinic acid (3-CQA), at doses of 4, 25 and 100 μg on plasma antioxidant capacity and plasma polyphenol content, measuring the differences before and after inducing a cerebral infarction in an experimental rat model. We compared them with 3-caffeoylquinic-free controls. The increase in total antioxidant capacity was only higher than in controls in 3-CQA treated animals with the highest dose. This increase in antioxidant capacity was not due to an increase in polyphenols. No differences between the experimental and control group were found regarding polyphenol content and cerebral infarction volume. In conclusion, this increase in antioxidant capacity in the group that received the highest dose of 3-CQA was not able to reduce experimental cerebral infarction.     

FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia

Background

The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/Principal Findings

FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/Significance

In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.     

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.     

Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.     

Messenger RNA coding for glutamine synthetase in cerebral hemispheres and astroglial cultures from mouse brain: A developmental study

Messenger RNAs from mouse brain hemispheres and from an enriched astroglial population of the same area were isolated, characterized and used to study Glutamine Synthetase (GS) processing during postnatal development using a m-RNA stimulated reticulocyte lysate system.The RNA preparations yielded distinct polypeptide products upon translation including high molecular weight species. Polypeptides in the range of 43–55 kDa appeared developmentally regulated in brain hemispheres but not in astroglia. After immunoprecipitation of the translation products with a GS antibody a major monomeric polypeptide was isolated on SDS/PAGE which migrated at the same position as the purified brain GS antigen (43 kDa).The translatable mRNA were optimal in the perinatal period and decreased until 300 days while GS-mRNA increased during the same period of time and closely paralleled the previously described GS activity profile in this brain area reaching an optimum at 15 days. Astroglial mRNAs were optimal at 18 days in vitro and decreased thereafter. The GS-mRNA was much lower in control astroglial cultures than in brain tissue, but in the presence of 10⁻⁶M hydrocortisone increased all over the growth period. The highest stimulation of GS-mRNA was observed at 18 days whereas the global mRNA decreased in the presence of the hormone.The GS-mRNAs from either 15-day-old brain hemispheres or 18-day in vitro hydrocortisone stimulated cultures were partially purified on a 5–30% linear sucrose gradient. Two GS-mRNAs, which sedimented respectively at 17S and 23S, were characterized. In addition, based on the profile of total proteins translated in vitro, we estimated that GS-mRNA constituted 0.01% of the brain hemisphere fraction and 0.3% in the astroglial hormone stimulated cells.     

Role of ICAM-1 (CD54) in the development of murine cerebral malaria

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.     

Low fertility increases descendant socioeconomic position but reduces long-term fitness in a modern post-industrial society

Adaptive accounts of modern low human fertility argue that small family size maximizes the inheritance of socioeconomic resources across generations and may consequently increase long-term fitness. This study explores the long-term impacts of fertility and socioeconomic position (SEP) on multiple dimensions of descendant success in a unique Swedish cohort of 14 000 individuals born during 1915–1929. We show that low fertility and high SEP predict increased descendant socioeconomic success across four generations. Furthermore, these effects are multiplicative, with the greatest benefits of low fertility observed when SEP is high. Low fertility and high SEP do not, however, predict increased descendant reproductive success. Our results are therefore consistent with the idea that modern fertility limitation represents a strategic response to the local costs of rearing socioeconomically competitive offspring, but contradict adaptive models suggesting that it maximizes long-term fitness. This indicates a conflict in modern societies between behaviours promoting socioeconomic versus biological success. This study also makes a methodological contribution, demonstrating that the number of offspring strongly predicts long-term fitness and thereby validating use of fertility data to estimate current selective pressures in modern populations. Finally, our findings highlight that differences in fertility and SEP can have important long-term effects on the persistence of social inequalities across generations.     

Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A (1)H-NMR and electronic absorbance study

The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N long left and right arrow X long left and right arrow D, where X represents a disordered globin structure (occurring at approximately 4 M urea) that still binds the heme.     

Targeted over-expression of glutamate transporter 1 (GLT-1) reduces ischemic brain injury in a rat model of stroke

Following the onset of an ischemic brain injury, the excitatory neurotransmitter glutamate is released. The excitotoxic effects of glutamate are a major contributor to the pathogenesis of a stroke. The aim of this study was to examine if overexpression of a glutamate transporter (GLT-1) reduces ischemic brain injury in a rat model of stroke. We generated an adeno-associated viral (AAV) vector expressing the rat GLT-1 cDNA (AAV-GLT1). Functional expression of AAV-GLT1 was confirmed by increased glutamate clearance rate in non-stroke rat brain as measured by in vivo amperometry. AAV-GLT1 was injected into future cortical region of infarction 3 weeks prior to 60 min middle cerebral artery occlusion (MCAo). Tissue damage was assessed at one and two days after MCAo using TUNEL and TTC staining, respectively. Behavioral testing was performed at 2, 8 and 14 days post-stroke. Animals receiving AAV-GLT1, compared to AAV-GFP, showed significant decreases in the duration and magnitude of extracellular glutamate, measured by microdialysis, during the 60 minute MCAo. A significant reduction in brain infarction and DNA fragmentation was observed in the region of AAV-GLT1 injection. Animals that received AAV-GLT1 showed significant improvement in behavioral recovery following stroke compared to the AAV-GFP group. We demonstrate that focal overexpression of the glutamate transporter, GLT-1, significantly reduces ischemia-induced glutamate overflow, decreases cell death and improves behavioral recovery. These data further support the role of glutamate in the pathogenesis of ischemic damage in brain and demonstrate that targeted gene delivery to decrease the ischemia-induced glutamate overflow reduces the cellular and behavioral deficits caused by stroke.     

Pilin C-terminal peptide binds asialo-GM1 in liposomes: a 2H-NMR study.

Wideline 2H-NMR observations are described demonstrating the interaction of a synthetic peptide (PAK), representing residues 128-144 of the binding domain of pilin surface protein from Pseudomonas aeruginosa, with a complex glycosphingolipid thought to be its natural receptor. The receptor glycolipid (asialo-GM1) carried 2H probe nuclei on the terminal and next-to-terminal carbohydrate residues and was present as a minor component in fluid phosphatidylcholine liposomes. The peptide induced spectral changes that could be understood as arising from receptor motional changes, without receptor immobilization on the NMR time scale of 10(4) s-1. Spectral effects were reversed by reduction of the single peptide disulfide bond--a structural feature previously shown to be a determinant of PAK conformation (Campbell AP, McInnes C, Hodges RS, Sykes BD. 1995. Biochemistry 34:16255-16268). This is the first demonstration of PAK interaction with its epithelial cell receptor in liposomes.     

Schistosoma co-infection protects against brain pathology but does not prevent severe disease and death in a murine model of cerebral malaria

Co-infections of helminths and malaria parasites are common in human populations in most endemic areas. It has been suggested that concomitant helminth infections inhibit the control of malaria parasitemia but down-modulate severe malarial disease. We tested this hypothesis using a murine co-infection model of schistosomiasis and cerebral malaria. C57BL/6 mice were infected with Schistosoma mansoni and 8-9 weeks later, when Schistosoma infection was patent, mice were co-infected with Plasmodium berghei ANKA strain. We found that a concomitant Schistosoma infection increased parasitemia at the beginning of the P. berghei infection. It did not protect against P. berghei-induced weight loss and hypothermia, and P. berghei-mono-infected as well as S. mansoni-P. berghei-co-infected animals showed a high case fatality between days 6 and 8 of malarial infection. However, co-infection significantly reduced P. berghei-induced brain pathology. Over 40% of the S. mansoni-P. berghei-co-infected animals that died during this period were completely protected against haemorrhaging, plugging of blood vessels and infiltration, indicating that mortality in these animals was not related to cerebral disease. Schistosoma mansoni-P. berghei-co-infected mice had elevated plasma concentrations of IL-5 and IL-13 and on day 6 lower levels of IFN-γ, IL-10, monocyte chemoattractant protein-1 (MCP-1) and monokine induced by IFN-γ (MIG) than P. berghei-mono-infected mice. We conclude that in P. berghei infections, disease and early death are caused by distinct pathogenic mechanisms, which develop in parallel and are differentially influenced by the immune response to S. mansoni. This might explain why, in co-infected mice, death could be induced in the absence of brain pathology.     

Conformational analysis of a segment in bacterioopsin by two-dimensional (1)H-NMR spectroscopy]

The spatial structure of a synthetic peptide, an analogue of the membrane spanning segment B (residues 34-65) of bacterioopsin from Halobacterium halobium, has been refined. Backbone torsion angles were derived from intensities of short-range interproton NOEs. These, together with a complete set of the NOEs integral intensities formed the basis for the three-dimensional structure refinement by the energy minimization with consideration of NOE penalty functions. Analysis indicates the right-handed alpha-helical conformation of segment B extending from Asp-38 to Tyr-64 with a kink of the helical axis (27 degrees) at Pro-50. The most stable region with an average root-mean-square deviation of 0.43 A between the backbone atoms includes residues 42-60 in six energy refined structures. The N-terminal part of segment B (residues 34-37) has no ordered conformation. The inferred structure is in close agreement with the electron cryomicroscopy structure of bacteriorhodopsin, differing from it in conformations of most of the side chains.