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Although the neurotoxic effects of homocysteine have been well elucidated, the effects of homocysteine in astrocytes have received little attention until recently. Previously we have demonstrated that elevated levels of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. However, the mechanisms behind such alterations remain unclear. As homocysteine is a key metabolite in one-carbon metabolism the present study examined if the effects of homocysteine on astrocyte function are mediated through an epigenetic mechanism. Following exposure to homocysteine for 72 h, global DNA methylation and H3K9 acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity and protein levels of DNA methyltransferase 3B were measured. Exposure to homocysteine resulted in global DNA hypomethylation (p0.05) and histone hyperacetylation (p0.05). Total DNA methyltransferase activity significantly decreased following exposure to homocysteine (from 11.5 ± 3.9 to 6.0 ± 1.7OD/h/mg protein, p0.01) which was accompanied by a significant reduction in protein levels of DNA methyltransferase 3B (p0.05). Treatment of astrocytes with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, mimicked the functional changes induced by homocysteine. In conclusion, the results demonstrate significant epigenetic modifications following exposure to homocysteine in astrocytes and these changes seem to mediate functional alterations.

Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.

J. Neurochem. (2010) 113, 735–748. Abstract Disruption of brain energy metabolism is the hallmark of cerebral ischemia, a major cause of death worldwide. Astrocytes play a key role in the regulation of brain metabolism and their vulnerability to ischemia has been described. Aiming to quantify the effects of an ischemic insult in astrocytic metabolism, primary cultures of astrocytes were subjected to 5 h of oxygen and glucose deprivation in a bioreactor. Flux distributions, before and after ischemia, were estimated by metabolic flux analysis using isotopic information and the consumption/secretion rates of relevant extracellular metabolites as constraints. During ischemia and early recovery, 30% of cell death was observed; several metabolic alterations were also identified reflecting a metabolic response by the surviving cells. In the early recovery (∼10 h), astrocytes up-regulated glucose utilization by 30% and increased the pentose phosphate pathway and tricarboxylic acid cycle fluxes by three and twofold, respectively. Additionally, a two to fivefold enhancement in branched-chain amino acids catabolism suggested the importance of anaplerotic molecules to the fast recovery of the energetic state, which was corroborated by measured cellular ATP levels. Glycolytic metabolism was predominant in the late recovery. In summary, this work demonstrates that changes in fluxes of key metabolic pathways are implicated in the recovery from ischemia in astrocytes.

This work investigates to which extent different carbon sources are metabolized and used for lipid biosynthesis in retrovirus producer cells, with the ultimate goal of understanding its importance regarding the stability/productivity of the vectors. For that purpose, isotopically labeled substrates (U- 13 C glucose, U- 13 C galactose, U- 13 C fructose, and U- 13 C glutamine) were used in combination with 13 C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry (GC–MS). Our results show that glucose plays the major role in lipid biosynthesis, whereas glutamine, fructose and galactose are not significantly incorporated into lipids. Moreover, a correlation between medium osmolality (imposed by the presence of sorbitol) and virus stability and productivity was verified, apparently due to an enhancement in sugar metabolism. Since low stability and short half-life constitute the major bottleneck in process development for retrovirus and other enveloped viral vectors, this work presents useful knowledge for improved process robustness for these essential gene therapy vectors.

The glutamate–glutamine cycle is an outstanding example of how essential neuronal–glial interactions are for brain function. For several decades, this and other metabolic cycles in the brain have only included neurons and astrocytes but not oligodendrocytes, the myelinating cells of the central nervous system (CNS). Recent data revealed that oligodendrocytes are highly metabolically active cells in the brain and, therefore, should not be ignored. Using 13C-labelled glucose in combination with nuclear magnetic resonance spectroscopy (MRS) and/or mass spectrometry (MS) it is possible to characterize metabolic functions in primary oligodendrocyte cultures. Mature rat oligodendrocytes avidly metabolize glucose in the cytosol and pyruvate derived from glucose in mitochondria. Moreover, they seem to have the ability of performing anaplerosis from pyruvate, which might enable them to synthesize metabolites de novo and transfer them to neighbouring cells. All these original findings highlight the importance of investigating oligodendrocyte metabolism separately from that of astrocytes and neurons to be able to discern the roles played by the individual partners. This is of particular importance in the white matter where the number of oligodendrocytes is considerable. The present book chapter provides some background on oligodendrocyte biology and physiology and summarizes the not very extensive information published on glucose metabolism in oligodendrocytes.

This work describes the development of a downstream process based upon membrane technology for the purification of Ehrlichia ruminantium (ER) elementary bodies, which can be used as an inactivated vaccine against heartwater for wild and domestic ruminants. Currently, ER purification is performed by a time consuming multistep centrifugation leading to a high level of host endothelial cell protein contamination. Herein, a simple and scaleable process based on depth filtration for clarification, and tangential flow filtration for concentration to effectively recover ER from infected endothelial cell microcarrier cultures is described. Specifically, depth filtration using 20 and 3 μm pore size membranes was applied to remove microcarriers from the bulk culture while tangential flow filtration was used to simultaneously remove additional cell debris and concentrating the ER to an appropriate level of volume reduction. The effects of transmembrane pressure and tangential filtration mode on ER purification were evaluated; three purification processes were compared to the commonly used centrifugation technique. Results showed that an ER recovery yield of 58% and volume reduction of 87% was achievable in less than 1 h of processing time when using membrane-based processes. This process enables a rapid purification of ER elementary bodies with a minimum of unit operations, reducing the overall cost of the vaccine production; similar approaches may be applied for the purification of other obligate intracellular bacteria with emerging impact on human and animal health.

Enhancing central nervous system (CNS) myelin regeneration is recognized as an important strategy to ameliorate the devastating consequences of demyelinating diseases such as multiple sclerosis. Previous findings have indicated that myelin proteins, which accumulate following demyelination, inhibit remyelination by blocking the differentiation of rat oligodendrocyte progenitor cells (OPCs) via modulation of PKCα. We therefore screened drugs for their potential to overcome this differentiation block. From our screening, tamoxifen emerges as a potent inducer of OPC differentiation in vitro. We show that the effects of tamoxifen rely on modulation of the estrogen receptors ERα, ERβ, and GPR30. Furthermore, we demonstrate that administration of tamoxifen to demyelinated rats in vivo accelerates remyelination. Tamoxifen is a well-established drug and is thus a promising candidate for a drug to regenerate myelin, as it will not require extensive safety testing. In addition, Tamoxifen plays an important role in biomedical research as an activator of inducible genetic models. Our results highlight the importance of appropriate controls when using such models.

Whereas astrocytes have been in the limelight on the metabolic glucose interaction scene for a while, oligodendrocytes are still waiting for a place. We would like to call oligodendrocyte interaction with astrocytes and neurons: NOA (neuron – oligodendrocyte – astrocyte) interactions. One of the reasons to find out more about oligodendrocyte interaction with neurons and astrocytes is to detect markers of healthy oligodendrocyte metabolism, to be used in diagnosis and treatment assessment in diseases such as Perinatal hypoxic-ischemic encephalopathy and multiple sclerosis in which oligodendrocyte function is impaired, possibly due to glutamate toxicity. Glutamate receptors are expressed in oligodendrocytes and also vesicular glutamate release in the white matter has received considerable attention. It is also important to establish if the glial precursor cells recruited to damaged areas are developing oligodendrocyte characteristics or those of astrocytes. Thus, it is important to study astrocytes and oligodendrocytes separately to be able to differentiate between them. This is of particular importance in the white matter where the number of oligodendrocytes is considerable. The present review summarizes the not very extensive information published on glucose metabolism in oligodendrocytes in an attempt to stimulate research into this important field.

Whereas astrocytes have been in the limelight of scientific interest in brain energy metabolism for a while, oligodendrocytes are still waiting for a place on the metabolic stage. We propose to term the interaction of oligodendrocytes with astrocytes and neurons: NOA (neuron–oligodendrocyte–astrocyte) interactions. One of the reasons to find out more about metabolic interactions between oligodendrocytes, neurons, and astrocytes is to establish markers of healthy oligodendrocyte metabolism that could be used for the diagnosis and assessment of white matter disease. The vesicular release of glutamate in the white matter has received considerable attention in the past. Oligodendrocyte lineage cells express glutamate receptors and glutamate toxicity has been implicated in diseases affecting oligodendrocytes such as hypoxic-ischaemic encephalopathy, inflammatory diseases and trauma. As oligodendrocyte precursor cells vividly react to injury it is also important to establish whether cells recruited into damaged areas are able to regenerate lost myelin sheaths or whether astrocytic scarring occurs. It is therefore important to consider metabolic aspects of astrocytes and oligodendrocytes separately. The present review summarizes the limited evidence available on metabolic cycles in oligodendrocytes and so hopes to stimulate further research interests in this important field.

Hypoglycaemia is characterized by decreased blood glucose levels and is associated with different pathologies (e.g. diabetes, inborn errors of metabolism). Depending on its severity, it might affect cognitive functions, including impaired judgment and decreased memory capacity, which have been linked to alterations of brain energy metabolism. Glucose is the major cerebral energy substrate in the adult brain and supports the complex metabolic interactions between neurons and astrocytes, which are essential for synaptic activity. Therefore, hypoglycaemia disturbs cerebral metabolism and, consequently, neuronal function. Despite the high vulnerability of neurons to hypoglycaemia, important neurochemical changes enabling these cells to prolong their resistance to hypoglycaemia have been described. This review aims at providing an overview over the main metabolic effects of hypoglycaemia on neurons, covering in vitro and in vivo findings. Recent studies provided evidence that non-glucose substrates including pyruvate, glycogen, ketone bodies, glutamate, glutamine, and aspartate, are metabolized by neurons in the absence of glucose and contribute to prolong neuronal function and delay ATP depletion during hypoglycaemia. One of the pathways likely implicated in the process is the pyruvate recycling pathway, which allows for the full oxidation of glutamate and glutamine. The operation of this pathway in neurons, particularly after hypoglycaemia, has been re-confirmed recently using metabolic modelling tools (i.e. Metabolic Flux Analysis), which allow for a detailed investigation of cellular metabolism in cultured cells. Overall, the knowledge summarized herein might be used for the development of potential therapies targeting neuronal protection in patients vulnerable to hypoglycaemic episodes.

The manufacture of enveloped virus, particularly retroviral (RV) and lentiviral (LV) vectors, faces the challenge of low titers that are aggravated under serum deprivation culture conditions. Also, the scarce knowledge on the biochemical pathways related with virus production is still limiting the design of rational strategies for improved production yields. This work describes the adaptation to serum deprivation of two human RV packaging cell lines, 293 FLEX and Te Fly and its effects on lipid biosynthetic pathways and infectious vector production. Total lipid content as well as cellular cholesterol were quantified and lipid biosynthesis was assessed by 13C-NMR spectroscopy; changes in gene expression of lipid biosynthetic enzymes were also evaluated. The effects of adaptation to serum deprivation in lipid biosynthesis were cell line specific and directly correlated with infectious virus titers: 293 FLEX cells faced severe lipid starvation—up to 50% reduction in total lipid content—along with a 68-fold reduction in infectious vector titers; contrarily, Te Fly cells were able to maintain identical levels of total lipid content by rising de novo lipid biosynthesis, particularly for cholesterol—50-fold increase—with the consequent recovery of infectious vector productivities. Gene expression analysis of lipid biosynthetic enzymes further confirmed cholesterol production pathway to be prominently up-regulated under serum deprivation conditions for Te Fly cells, providing a genotype–phenotype validation for enhanced cholesterol synthesis. These results highlight lipid metabolism dynamics and the ability to activate lipid biosynthesis under serum deprivation as an important feature for high retroviral titers. Mechanisms underlying virus production and its relationship with lipid biosynthesis, with special focus on cholesterol, are discussed as potential targets for cellular metabolic engineering. Biotechnol. Bioeng. 2012; 109:1269–1279. © 2011 Wiley Periodicals, Inc.

The development of serum-free processes is crucial for the safety and standardization of biopharmaceuticals production. However, serum replacement is still a challenge in several situations, namely the manufacturing of enveloped viruses such as recombinant retroviral vectors (RV). With the main goal of understanding the relationship between RV production and serum, the effect of serum deprivation was analyzed in terms of cell growth, metabolic behavior, productivity and vector characteristics using two different human producer cell lines: 293 FLEX 18 and Te Fly Ga 18. The reduction of serum in the culture media resulted in pronounced decreases in infectious vector titer (I.P.), although the titer of total produced vectors (T.P.) was maintained. Lipids, particularly cholesterol, were identified as the main serum component necessary for I.P. production, confirmed by using delipidated serum. Culture media supplementation with lipids resulted in a 9-fold cell productivity increment, allowing the total recovery of viral titers and also an additional 4-fold increment. The characterization of cell lipid content using 13C-NMR spectroscopy revealed that long-term serum deprivation can induce significant increases in lipid biosynthetic capacity with the concomitant recovery of I.P. production. This behavior was, however, cell line dependent. Producer cells lipid metabolism was, thus, identified as a key parameter for the ability of cells to adapt to serum deprivation. Furthermore, it was confirmed to be a potential target for improving product quality as the use of lipid supplements allowed for an improvement in I.P/T.P ratio of more than one order of magnitude.

Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.

Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.

Metabolic models have been used to elucidate important aspects of brain metabolism in recent years. This work applies for the first time the concept of isotopic transient 13C metabolic flux analysis (MFA) to estimate intracellular fluxes in primary cultures of astrocytes. This methodology comprehensively explores the information provided by 13C labeling time-courses of intracellular metabolites after administration of a 13C-labeled substrate. Cells were incubated with medium containing [1-13C]glucose for 24 h and samples of cell supernatant and extracts collected at different time points were then analyzed by mass spectrometry and/or high performance liquid chromatography. Metabolic fluxes were estimated by fitting a carbon labeling network model to isotopomer profiles experimentally determined. Both the fast isotopic equilibrium of glycolytic metabolite pools and the slow labeling dynamics of TCA cycle intermediates are described well by the model. The large pools of glutamate and aspartate which are linked to the TCA cycle via reversible aminotransferase reactions are likely to be responsible for the observed delay in equilibration of TCA cycle intermediates. Furthermore, it was estimated that 11% of the glucose taken up by astrocytes was diverted to the pentose phosphate pathway. In addition, considerable fluxes through pyruvate carboxylase [PC; PC/pyruvate dehydrogenase (PDH) ratio = 0.5], malic enzyme (5% of the total pyruvate production), and catabolism of branched-chained amino acids (contributing with ∼40% to total acetyl-CoA produced) confirmed the significance of these pathways to astrocytic metabolism. Consistent with the need of maintaining cytosolic redox potential, the fluxes through the malate–aspartate shuttle and the PDH pathway were comparable. Finally, the estimated glutamate/α-ketoglutarate exchange rate (∼0.7 μmol mg prot−1 h−1) was similar to the TCA cycle flux. In conclusion, this work demonstrates the potential of isotopic transient MFA for a comprehensive analysis of energy metabolism. Copyright: © 2011 Amaral, Teixeira, Håkonsen, Sonnewald and Alves. This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.

Metabolic models have been used to elucidate important aspects of brain metabolism in recent years. This work applies for the first time the concept of isotopic transient 13C metabolic flux analysis (MFA) to estimate intracellular fluxes in primary cultures of astrocytes. This methodology comprehensively explores the information provided by 13C labeling time-courses of intracellular metabolites after administration of a 13C-labeled substrate. Cells were incubated with medium containing [1-13C]glucose for 24 h and samples of cell supernatant and extracts collected at different time points were then analyzed by mass spectrometry and/or high performance liquid chromatography. Metabolic fluxes were estimated by fitting a carbon labeling network model to isotopomer profiles experimentally determined. Both the fast isotopic equilibrium of glycolytic metabolite pools and the slow labeling dynamics of TCA cycle intermediates are described well by the model. The large pools of glutamate and aspartate which are linked to the TCA cycle via reversible aminotransferase reactions are likely to be responsible for the observed delay in equilibration of TCA cycle intermediates. Furthermore, it was estimated that 11% of the glucose taken up by astrocytes was diverted to the pentose phosphate pathway. In addition, considerable fluxes through pyruvate carboxylase [PC; PC/pyruvate dehydrogenase (PDH) ratio = 0.5], malic enzyme (5% of the total pyruvate production), and catabolism of branched-chained amino acids (contributing with ∼40% to total acetyl-CoA produced) confirmed the significance of these pathways to astrocytic metabolism. Consistent with the need of maintaining cytosolic redox potential, the fluxes through the malate–aspartate shuttle and the PDH pathway were comparable. Finally, the estimated glutamate/α-ketoglutarate exchange rate (∼0.7 μmol mg prot−1 h−1) was similar to the TCA cycle flux. In conclusion, this work demonstrates the potential of isotopic transient MFA for a comprehensive analysis of energy metabolism.

Although glucose is the primary cerebral fuel, the brain is able to metabolize other substrates in hypoglycemia. Nevertheless, the metabolic consequences of this pathology at the cellular level remain largely unknown. Taking advantage of the metabolic flux analysis (MFA) methodology, this work was aimed at investigating and quantifying the effects of hypoglycemia on cerebellar neurons. After 12 hr without glucose, primary cultures were incubated with medium containing [1,6-(13)C]glucose and unlabeled glutamine, and metabolism was monitored for 30 hr. Metabolic rates of glucose, lactate, and amino acids were determined based on cell supernatant analysis and used to estimate metabolic fluxes with MFA. Percent (13)C enrichment time profiles of different keto and amino acids were measured by mass spectrometry in cell extracts and compared with the MFA results. Hypoglycemia decreased the glucose uptake rate and glycolytic metabolism by 35% whereas glutamine uptake was increased fourfold. Flux estimations fit well with data from (13)C labeling dynamics, indicating a significant activation of the pyruvate recycling (PR) pathway, accounting for 43% of the total pyruvate synthesized under control conditions and up to 71% after hypoglycemia. Increased PR appeared to be due mainly to increased glutamine oxidation given the higher label dilution observed in the hypoglycemia group. In summary, this work provides new evidence for PR as an important pathway for glutamine oxidation in cerebellar neurons, particularly after glucose deprivation.

The work presented herein reports the improvement achieved on endothelial cell culture conditions and the production of the Rickettsiales Ehrlichia ruminantium (ER) using stirred tanks towards the development of a cost effective process for the widespread application of the inactivated vaccine against heartwater. After the selection of the best endothelial cell line (according to the maximum cell concentration), ER production in stirred tanks was evaluated. Key parameters related with the infection strategy such as serum concentration and multiplicity of infection were then analyzed. The results showed that it was possible to produce ER in stirred tank bioreactors and that the production yields can be increased by a factor of 6 using a serum-free medium during and after the infection process. To expand the process and improve ER production yields, ER growth kinetics was characterized showing that this stirring culture system is capable of efficient bacterial amplification with maximum titers going up to 2.2 log10ER/cell. A downstream processing for the purification of ER is also described, taking into account ER recovery yields, the number of steps and endothelial protein contamination. To ensure the successful use of this vaccine, stability tests were also performed with special emphasis on the vaccine formulation and storage temperature; the potency of the final vaccine was validated with test trials in animals (goats). Overall, these results present an attractive process for the production/purification of obligate intracellular bacteria with economical impact in animal and human health, using robust technologies such as stirred tank, microcarriers and membranes.

A metabolic flux analysis (MFA) study was performed to infer about the metabolic plasticity of astrocytes when exposed to two environmental stresses. In the first study an astrocytic culture was subjected to a hypoglycemic period of 24 h and the metabolic response was investigated during and after the insult. The second study involved another astrocytic culture that was subjected to an anoxic episode of three hours; and the metabolism was evaluated both before/during anoxia and in the recovery period (normal O2 levels re-established). In both studies [1-13C]glucose was administrated to the cultures and the incorporation of the label into metabolites was analysed by nuclear magnetic resonance (NMR) spectroscopy, allowing the assessment of particular pathways used by astrocytes. The oxidative metabolism of astrocytes exposed to hypoglycaemia increased during the recovery phase since an increase in tricarboxylic acid (TCA) cycle fluxes was observed. During anoxia, astrocytes respond by up-regulating their glycolytic capacity in order to maintain their energy requirements. When the O2 levels were restored, a metabolic shift towards a more oxidative utilization of glucose was observed when compared to pre-anoxic conditions.

This work evaluates the effect of purification process and storage conditions (buffer formulation and temperature) on the efficacy of Ehrlichia ruminantium (ER) elementary bodies to be used as an inactivated vaccine candidate against heartwater. In vitro assays revealed that, to avoid major losses in ER integrity and corresponding antigenic properties, a buffer with pH between 5.6 and 8 and an osmolality above 100 mOsmol/kg H2O is recommended. Amongst the tested formulations, both PBS and NaCl have shown to stabilize ER antigens at -20°C. To assess the protective properties of the different vaccine formulations, in vivo experiments were performed using a goat model. The results obtained showed that the preparation of ER antigens using a novel membrane-based purification strategy and a simple vaccine formulation (NaCl, -20°C) induced equivalent protection to the conventional vaccine based on ER antigens prepared by a multistep centrifugation methodology and stored at -20°C in PBS buffer. © 2007 Elsevier Ltd. All rights reserved.